Production of vanillin

ABSTRACT

A method of producing vanillin comprising the steps of: (1) providing trans-ferulic acid or a salt thereof; and (2) providing trans-ferulate: CoASH ligase activity (enzyme activity I), trans-feruloyl ScoA hydratase activity (enzyme activity II), and 4-hydroxy-3-methoxyphenyl-β-hydroxy-propionyl SCoA (HMPHP SCoA) cleavage activity (enzyme activity III). Conveniently the enzymes are provided by  Pseudomonas fluorescens  Fe3 or a mutant or derivative thereof. Polypeptides with enzymes activities II and III and polynucleotides encoding said polypeptides. Use of said polypeptides or said polynucleotides in a method for producing vanillin.

[0001] The present invention relates principally to the production ofvanillin (4-hydroxy-3-methoxybenzaldehyde), particularly to theproduction of vanillin other than by extraction from the Vanilla pod.

[0002] Vanillin is an important food and drink flavouring agent and amajor flavour component of natural vanilla from the Vanilla pod. The useof natural vanilla is limited by its high price. Synthetic vanillin,commonly derived from sulphite liquors produced during the processing ofwood pulp for paper manufacture, is frequently used as a low-costvanilla substitute. Alternative biological processes for the productionof natural vanillin and allied flavourings would have considerableindustrial value and utility, most particularly if such processes couldfacilitate the production of vanillin and/or allied flavourings directlyin a fermented food or beverage.

[0003] The mechanism of vanillin biosynthesis in Vanilla remainssubstantially uncharacterised. M. H. Zenk (Anal. Z. Pflanzenphysiol 53,404-414 (1965)) showed that vanillin was derived from trans-ferulate(4-hydroxy-3-methoxy-trans-cinnamate) and proposed a mechanism analogousto the classical β-oxidation of fatty acids, with cleavage of a β-ketothioester to produce acetyl SCoA and vanilloyl SCoA(4hydroxy-3-methoxybenzoyl SCoA) and subsequent reduction and CoASHrelease to generate vanillin. C. Funk and P. E. Brodelius (PlantPhysiol. 94, 95-101; 102-108 (1990); 99, 256-262 (1992)), proposed adifferent route, in which the 4-hydroxy group of trans-ferulate becamesuccessively methylated and demethylated during the pathway of vanillinbiosynthesis; however, the detailed enzymology was not elucidated. Inpotato tubers and in the fungus, Polyporus hispidus (C. J. French, C. P.Vance and G. H. N. Towers, Phytochemistry 15, 564-566 (1976)), in cellcultures of Lithospermum erythrorhizon (K. Yazaki, L. Heide and M.Tabata, Phytochemistry 30, 2233-2236 (1991)) and in cell cultures ofcarrot (J.-P. Schnitzler, J. Madlung, A. Rose and H. U. Seitz, Planta188, 594-600 (1992)), evidence was obtained from in vitro studies thatthe corresponding analogue of vanillin, 4-hydroxybenzaldehyde, was anintermediate in the formation of 4-hydroxybenzoate from 4-coumarate(4-hydroxy-trans-cinnamate). There was no requirement for ATP or CoASH,thus apparently ruling out a β-oxidation mechanism. Further studies withcell-free extracts of Lithospermum erythrorhizon, however, have incontrast recently established the presence of a β-oxidation route forthe conversion of 4-coumarate to 4-hydroxybenzoate (R. Löscher and L.Heide, Plant Physiol. 106, 271-279 (1994)); in this case, the conversionwas dependent on ATP, Mg²⁺ ions and NAD⁺ and proceeded via4-hydroxybenzoyl SCoA, without the intermediate formation of4-hydroxybenzaldehyde.

[0004] In the Gram-negative bacterium, Pseudomonas acidovorans,trans-ferulate was shown to be catabolised to vanillate and acetate,apparently via vanillin (A. Toms and J. M. Wood, Biochemistry 9, 337-343(1970)). Although in cell-free extracts NAD⁺ was necessary for theoxidation of vanillin to vanillate and for the further oxidation ofvanillate to protocatechuate and formate, no mention was made of anyother cofactor requirements. Further studies of ferulate utilisation inPseudomonas species have been reported (V. Andreoni and G. Bestetti,FEMS Microbiology Ecology 53, 129-132 (1988); T. Omori, K. Hatakeyamaand T. Kodama, Appl. Microbiol. Biotechnol. 29, 497-500 (1988); Z.Huang, L. Dostal and J. P. N. Rosazza, Appl. Env. Microbiol. 59,2244-2250 (1993)); however, these have not sought to elucidate furtherthe mechanism of the two-carbon cleavage of ferulate. Zenk et al (1980)Anal. Biochem. 101, 182-187 describe a procedure for the enzymaticsynthesis and isolation of cinnamoyl-CoA thioesters using a bacterialsystem. In contrast, the enzymology and genetics of the utilisation ofsimple benzene derivatives, including benzoic acids and phenols, byPseudomonas have been intensively studied (T. K. Kirk, T. Higuchi andH.-M. Chang (eds.), “Lignin biodegradation”, CRC Press, Boca Raton, Fla,USA (1980); D. T. Gibson (ed.), “Microbial degradation of organiccompounds”, Marcel Dekker, New York (1984); J. L. Ramos, A.Wasserfallen, K. Rose and K. N. Timmis, Science 235, 593-596 (1987); C.S. Harwood, N. N. Nichols, M. K. Kim, J. L. Ditty and R. E. Parales, J.Bacteriol. 176, 6479-6488 (1994); S. Romerosteiner, R. E. Parales, C. S.Harwood and J. E. Houghton, J. Bacteriol. 176, 5771-5779 (1994); J.Inoue, J. P. Shaw, M. Rekik and S. Harayama, J. Bacteriol. 177,1196-1201 (1995)).

[0005] A survey of potential microbial routes to aromatic aldehydes,including routes (i) from trans-cinnamic acids, (ii) from benzoic acidsby reduction and (iii) by conversion of aromatic amino acids tophenylpyruvic acids followed by treatment with base, has been presentedby J. Casey and R. Dobb (Enzyme Microb. Technol. 14, 739-747 (1992)).

[0006] U.S. Pat. No. 5,128,253 describes a method of producing vanillinfrom ferulic acid by various microorganisms and extracts thereof orenzymes derived therefrom in the presence of a sulphydryl compound butdoes not disclose what any of the enzymes involved in the conversion offerulic acid to vanillin are. U.S. Pat. No. 5,279,950 is acontinuation-in-part application of U.S. Pat. No. 5,128,253 whichadditionally describes that Vanilla calluses can be used in the process.

[0007] WO 94/13614 describes the production of vanillin from ferulicacid by the action of Vanilla root material and makes use of anadsorbent, such as charcoal, to extract vanillin but does not disclosethe specific enzymes involved.

[0008] EP 0 453 368 describes that a culture of Pycnoporus can converttrans-ferulic acid into vanillin but does not disclose the specificenzymes involved.

[0009] WO 94/02621 describes the production of vanillin fromtrans-ferulic acid by the action of a lipoxygenase enzyme. EP 0 405 197describes the production of vanillin from eugenol/isoeugenol by bacteriafrom the genera Serratia, Klebsiella and Enterobacter by oxidation.

[0010] Vanillin may also be produced from phenolic stilbenes as ismentioned in Hagedorn & Kaphammer (1994) Ann. Rev. Microbiol. 48,773-800.

[0011] Vanillic acid is also a useful compound as it can be polymerisedinto oligomers or used as a monomer in the synthesis of polyesters;similarly p-hydroxybenzoic acid is also useful for polymer synthesis.

[0012] A first aspect of the invention provides a method of producingvanillin comprising the steps of

[0013] (1) providing trans-ferulic acid or a salt thereof; and

[0014] (2) providing trans-ferulate:CoASH ligase activity (enzymeactivity I), trans-feruloyl ScoA hydratase activity (enzyme activityII), and 4-hydroxy-3-methoxyphenyl-β-hydroxy-propionyl SCoA (HMPHP SCoA)cleavage activity (enzyme activity III).

[0015] The advantages of the present invention over chemical synthesisor extraction from the Vanilla pod include (i) economic advantage overextraction from Vanilla pod and freedom from geographical dependence onVanilla growing areas; (ii) the ability to produce vanillin by a naturalprocess, involving biological catalysts; (iii) the benefits ofgenerating a natural flavour in situ in a fermented food or beverage, ifthe genes are expressed in appropriate food-grade hosts—eg lactic acidbacteria or yeasts; and (iv) the possibility of expanding the range ofplants in which vanillin and related substances might be produced andfrom which they might be extracted. These and other examples of themethods of the invention are described in more detail below.

[0016] We have determined the mechanism of chain-shortening oftrans-ferulate (trans-ferulic acid) by a strain of Pseudomonasfluorescens (named Ps. fluorescens biovar. V, strain AN103 and which wehave abbreviated at some points to AN103) isolated from soil. Our dataindicate clearly that vanillin (4-hydroxy-3-methoxy benzaldehyde) is anintermediate and that the mechanism does not involve β-oxidation. Thevanillin pathway of Ps. fluorescens biovar. V, strain AN103 is describedin FIG. 1. Trans-ferulic acid (or a salt thereof) is interconverted withtrans-feruloyl ScoA in the presence of CoASH; trans-feruloyl SCoA isinterconverted with 4-hydroxy-3-methoxyphenyl-β-hydroxypropionyl SCoA(HMPHP SCoA); and HMPHP SCoA is interconverted with vanillin.

[0017] For convenience, trans-ferulate:CoASH ligase activity is calledenzyme activity I, trans-feruloyl SCoA hydratase activity is calledenzyme activity II; and HMPHP SCoA cleavage activity is called enzymeactivity III. The interconversions performed by these enzyme activitiesis shown in FIG. 1.

[0018] The method of producing vanillin provided by the inventiontherefore includes the steps of exposing trans-ferulic acid or a saltthereof to enzyme activity I and forming a product, exposing the saidproduct of enzyme activity I to enzyme activity II to form a product andexposing the said product of enzyme activity II to enzyme activity IIIto form a product.

[0019] Trans-ferulic acid or a salt thereof may be provided directly,for example by supplying pre-prepared trans-ferulic acid or a saltthereof, or it may be provided indirectly, for example by supplying aprecursor of trans-ferulic acid or a precursor of a salt oftrans-ferulic acid and means to convert the said precursor intotrans-ferulic acid or a salt thereof. As is described in more detailbelow, it is convenient if the precursor is an ester of trans-ferulicacid and the means to convert said ester is a suitable esterase. By“providing trans-ferulate:CoASH ligase activity (enzyme activity I),trans-feruloyl ScoA hydratase activity (enzyme activity II), and4-hydroxy-3-methoxyphenyl-β-hydroxy-propionyl SCoA (HMPHP ScoA) cleavageactivity (enzyme activity III)” we include the provision of the enzymeactivities in any suitable form to effect the said production ofvanillin. For example, as is discussed in more detail below, the methodof the invention specifically includes, but is not limited to, theprovision of the enzyme activities (a) by intact or permeabilised Ps.fluorescens biovar. V, strain AN103 or a mutant thereof, (b) at leastone of enzyme activities II or III of which is in a form substantiallyfree of cellular material, (c) by intact or permeabilised cells inculture, particularly microorganisms, which have been geneticallymodified to contain genes which encode enzyme activities II or III (forexample, food grade microorganisms such as lactic acid bacteria andbrewing yeast), and (d) by plants which have been genetically modifiedto contain genes which encode said enzyme activities.

[0020] It is preferred if means for converting vanillin to anon-vanillin product is absent or reduced. Of course, the enzymeactivity III is not such a means. Conveniently, these enzyme activitiesare provided by the soil bacterium Pseudomonas fluorescens biovar. V,strain AN103 the said bacterium being that deposited under the BudapestTreaty at the National Collection of Industrial and Marine BacteriaLimited, AURIS Business Centre, 23 St. Machar Drive, Aberdeen AB2 1RY,Scotland under Accession No NCIMB 40783, or a mutant or variant thereof.By “mutant or variant thereof” we include any mutant or variant of thesaid bacterium provided that the bacterium retains the said enzymeactivities whether or not at the same levels. It will be appreciatedthat the said enzyme activities can be retained even if the genesencoding said enzymes are mutated. For example, mutants whichconstitutively express (as opposed to conditionally or induciblyexpress) the said enzyme activities are particularly useful mutants ofPs. fluorescens biovar. V, strain AN103, as are variants in which one ormore of the enzymes with the said activities exhibit more favourablekinetic characteristics (for example, an increased turnover number or adecreased K_(m))

[0021] When Ps. fluorescens biovar. V, strain AN103 is growing ontrans-ferulate it will derive maximum benefit if vanillin is catabolisedfurther in order to provide more energy. However, in order to maximisethe production of vanillin by Ps. fluorescens biovar. V, strain AN103 itis preferred that the means for converting vanillin to a non-vanillinproduct is absent or reduced. We have found that in Ps. fluorescensbiovar. V, strain AN103 vanillin is converted to vanillic acid or a saltthereof by vanillin:NAD⁺ oxidoreductase. It is preferred if a mutant ofPs. fluorescens biovar. V, strain AN103 wherein the vanillin:NAD⁺oxidoreductase activity is absent or reduced is used in the method. Sucha mutant can be made using a gene replacement strategy with a disruptedvanillin:NAD oxidoreductase gene, or a sequence of DNA from which thisgene has been deleted. Gene replacement is well known in the art ofbacterial genetics. Alternatively, isolation of such a mutant may beachieved by classical chemical mutagenesis, selecting on the basis ofinability to grow on vanillin.

[0022] It will be appreciated that there are other means for convertingvanillin to a non-vanillin product and it is preferred if these areabsent or reduced in the method.

[0023] Although Ps. fluorescens biovar. V, strain AN103 or mutants orvariants thereof themselves are useful in the method of the invention aswhole cells or permeabilised or immobilised cells, it is preferred ifthe enzyme activities I, II and III are provided by an intact-cell-freesystem of Ps. fluorescens biovar, V, stain AN103 or a mutant or variantthereof. Suitable systems and extracts may be used by methods well knownin the art, for example by French pressure cell or sonication followedby centrifugation. Alternatively, whole cells may be permeabilised usingmethods well known in the art, for example using detergents such asdimethyl sulphoxide (DMSO).

[0024] Using such an intact-cell-free system allows the necessarysubstrates and any cofactors to reach readily the relevant enzymes andfor the products to be released readily into the reaction medium if thisis necessary for further reaction; however as discussed below, at leastsome of the enzymes of the invention may be involved in substrate(metabolic) channelling.

[0025] We have found that none of the enzyme activities I, II and IIIfrom Ps. fluorescens biovar. V, strain AN103 is dependent on NAD⁺whereas enzyme activity IV from Ps. fluorescens (vanillin:NAD⁺oxido-reductase) requires NAD⁺.

[0026] Thus, a preferred way of reducing means for converting vanillinto a non-vanillin product in an intact-cell-free system of Ps.fluorescens biovar. V, strain AN103 (or in a cell-permeabilised systemof Ps. fluorescens biovar. V, strain AN103) is to omit NAD⁺ from thereaction system. Any exogenous NAD⁺ is readily and rapidly depleted bythe presence of trans-ferulate in the system.

[0027] For the microorganisms of the present invention which can be usedin the method of vanillin production, including Ps. fluorescens biovar.V, strain AN103, at least three main types of bioreactor may be used forthe biotransformation reactions: the batch tank, the packed bed and thecontinuous-flow stirred tank; their applications and characteristicshave been reviewed (M. D. Lilly in “Recent Advances in Biotechnology”,eds. F. Vardar-Sukan and S. S. Sukan, Kluwer Academic Publishers,Dordrecht, 1992, pp 47-68 and loc. cit.).

[0028] As is described in more detail below, enzyme activities II andIII are available free from other enzyme activities, for exampledirectly or indirectly from Ps. fluorescens biovar. V, strain AN103 andfrom other organisms or cells which have been genetically modified toexpress genes encoding the said enzyme activities.

[0029] It will be appreciated that other microorganisms will be foundwhich will be useful in the methods of the invention, for example, byscreening. Such microorganisms and methods of screening and methods ofuse form part of the invention. The method of screening for othermicroorganisms possessing activities I, II and III is essentially thatalready described in the Materials and Methods section in the Examplesfor the isolation of AN 103. The important aspect is isolation from anenvironment rich in trans-ferulate or related compounds (eg4-trans-coumarate, trans-caffeate [3,4-dihydroxy-trans-cinnamate] which,as described below, may also be substrates for enzyme activity I) andselection for growth on trans-ferulate (preferably) as sole carbonsource. In practice, preferred sources are those in which plant-derivedmaterials are being degraded; in addition to soil or compost, this wouldinclude the outflow or residues from factories or other installationsprocessing such materials—eg sugar-beet factories, cocoa fermentationheaps etc—and the contents of the gastro-intestinal tract, particularlyof ruminants and other herbivores. It is possible that anaerobes mightbe found possessing these activities and due account can readily betaken of this in the isolation procedure. A priori, isolation oforganisms with these activities might also be possible from marineenvironments.

[0030] Genera in which further microorganisms useful in the inventionwill be found include Pseudomonas, Arthrobacter, Alcaligenes,Acinetobacter, Flavobacterium, Agrobacterium, Rhizobium, Streptomyces,Saccharomyces, Penicillium and Aspergillus.

[0031] An alternative or additional approach is to use any one of thePseudomonas genes encoding enzyme activities II or III described hereinor redundant sequences designed from the Pseudomonas enzyme amino-acidsequences in DNA probes or PCR amplification strategies to find relatedgenes in other organisms. As is made more clear below, enzymes andnucleotide sequences which are functionally equivalent to those ofisolated from AN103 but which differ in sequence form part of theinvention.

[0032] Our studies indicate that the enzyme which interconvertstrans-ferulate and trans-feruloyl SCoA in Ps. fluorescens biovar. V,strain AN103 uses Coenzyme A (CoASH), ATP and Mg²⁺ or other functionallyequivalent cofactors. Thus, it is preferred that the method furthercomprises the step of (3) providing any one of the cofactors CoASH, ATPor Mg²⁺ or other functionally equivalent cofactors. ATP is adenosinetriphosphate. It is well known that other functionally equivalentcofactors can substitute in some cases for CoASH, ATP or Mg²⁺. Forexample Mn²⁺ may be used in place of Mg²⁺ and derivatives or analoguesof ATP, preferably with a hydrolysable γ-phosphate, may be used in placeof ATP.

[0033] We have also determined that, at least when the enzyme activity Iis provided by the Pseudomonas AN103 enzyme which interconvertstrans-ferulate and trans-feruloyl SCoA and which enzyme uses ATP andCoenzyme ASH, it is convenient to include a system wherein either one,or both, of the cofactors Coenzyme ASH and ATP is recycled. Thefollowing ATP generation and CoASH recycling systems are preferred.

[0034] ATP generation:-

[0035] (i) trans-Ferulate+CoASH+ATP0+H₂O→Vanillin+Acetyl SCoA+AMP+PPi(overall reaction catalysed by Ps. fluorescens biovar. V, strain AN103extract)

[0036] (ii) AMP+ATP⇄2 ADP (adenylate kinase)

[0037] (iii) Acetyl˜P+ADP⇄Acetate+ATP (acetate kinase)

[0038] (iv) Sum: trans-Ferulate+CoASH+2 Acetyl˜P+H₂O→Vanillin+AcetylSCoA+2 Acetate+PPi

[0039] CoASH recycling is achievable using commercially-availablecitrate synthase (EC 4.1.3.7) and citrate lyase (EC 4.1.3.6), viz:-

[0040] (v) Acetyl SCoA+Oxaloacetate+H₂O⇄Citrate+CoASH (citrate synthase)

[0041] (vi) Citrate⇄Oxaloacetate+Acetate (citrate lyase)

[0042] Overall sum, (iv)-(vi):

[0043] (vii) trans-Ferulate+2 Acetyl˜P+2 H₂O→Vanillin+3 Acetate+PPi

[0044] The acetyl˜P used in the overall process, (vii), would not itselfbe generated by enzymic means; however, none of its atoms would appearin the vanillin product.

[0045] Acetyl phosphate is commercially available or can be synthesisedusing the method described by Stadtman (1957) Meth. Enzymol. 3, 228-231.

[0046] The reagents are commercially available from, for example SigmaChemical Co, Fancy Road, Poole, Dorset, UK. Citrate lyase is typicallyfrom Enterobacter aerogenes; citrate synthase is typically from chickenheart, pigeon breast muscle or porcine heart.

[0047] Thus, it is preferred if coenzyme ASH is recycled using theenzymes citrate synthase and citrate lyase; and it is preferred if theATP is generated using the enzymes adenylate kinase (EC 2.7.4.3) andacetate kinase (EC 2.7.2.1).

[0048] The co-factor recycling system is particularly preferred whenusing an intact-cell-free system.

[0049] Trans-ferulic acid or a salt thereof is readily available fromplant material. Suitably, trans-ferulic acid or a salt thereof isreleased from the plant material by the action of ferulic acid esterase.Thus, in a particularly preferred embodiment of the invention thetrans-ferulic acid or salt thereof is provided by the action of ferulicacid esterase on plant material.

[0050] Trans-ferulic acid and trans4-coumaric acid can togetherrepresent up to 1.5% by weight of the cell walls of temperate grasses(R. D. Hartley and E. C. Jones, Phytochemistry 16, 1531-1534 (1977)).Trans-ferulic acid is reported to comprise 0.5% (w/w) of wheat bran (M.C. Ralet, J.-F. Thibault and G. Della Valle, J. Cereal Sci. 11, 249-259(1990)), 3.1% of maize bran (L. Saulnier, C. Marot, E. Chanliaud andJ.-F. Thibault, Carbohydr. Polym. 26, 279-287 (1995)) and 0.8% of sugarbeet pulp (V. Micard, G. M. G. C. Renard and J.-F. Thibault,Lebensm.-Wiss. u-Technol. 27, 59-66 (1994)). These materials are amongstthe preferred sources of trans-ferulic acid. Since trans-ferulic acid ispresent esterified with cell-wall polysaccharides, hydrolysis isessential. Alkaline or acid hydrolysis is possible, but enzymichydrolysis is preferred. Typically, the initial step is the partialenzymic hydrolysis of carbohydrates (arabinans, xylans,rhamnogalacturanans) to which trans-ferulate is linked, followed by therelease of trans-ferulate from the oligosaccharide fragments bytrans-ferulic acid esterase activity. In practice, both steps may occursimultaneously in the reaction mixture. Descriptions of representativelaboratory-scale processes are available in the literature (for examplesee L. P. Christov and B. A. Prior, Enzyme Microb. Technol. 15, 460-475(1993)); C. B. Faulds and G. Williamson, Appl. Microbiol. Biotechnol.43, 1082-1087 (1995); C. B. Faulds, P. A. Kroon, L. Saulnier, J.-F.Thibault and G. Williamson, Carbohydrate Polymers 27, 187-190 (1995)).Phenolic acid-releasing enzymes have been reported from a number ofmicroorganisms, including Streptomyces olivochromogenes (C. B. Fauldsand G. Williamson, J. Gen. Microbiol. 137, 2337-2345 (1991)),Penicillium pinophilum (A. Castanares, S. I. McCrae and T. M. Wood,Enzyme Microb. Technol. 14, 875-884 (1992)), Neocallimastix spp. (W. S.Borneman, R. D. Hartley, W. H. Morrison, D. E. Akin and L. G. Ljungdahl,Appl. Microbiol. Biotechnol. 33, 345-35,1 (1990)), Schizophyllum commune(R. C. MacKenzie and D. Bilous, Appl. Envir. Microbiol. 54, 1170-1173(1988)) and Aspergillus spp. (M. Tenkanen, J. Schuseil, J. Puls and K.Poutanen, J. Biotechnol. 18, 69-84 (1991); C. B. Faulds and G.Williamson, Microbiology 140, 779-787 (1994)). A trans-ferulic acidesterase (XYLD) has been characterised from Pseudomonas fluorescenssubsp. cellulosa, together with an arabinofuranosidase (XYLC) and anendoxylanase (XYLB; L. M. A. Ferreira, T. M. Wood, G. Williamson, C. B.Faulds, G. P. Hazlewood and H. J. Gilbert, Biochem. J. 294, 349-355(1993)). The genes for all three enzymes have been isolated (G. P.Hazlewood and H. J. Gilbert, in “Xylans and Xylanases”, eds. J. Visser,G. Beldman, M. A. Kusters-van Someren and A. G. J. Voragen, Elsevier,Amsterdam, pp 259-273 (1992)). All of these references are incorporatedherein by reference.

[0051] Thus, advantageously the trans-ferulic acid or a salt thereof maybe provided by the action of trans-ferulic acid esterase on said ester.More particularly, it is advantageous to introduce a gene encoding saidesterase into a host cell or organism which is being used in the methodsof the invention. Thus, it is convenient to introduce a trans-ferulicacid esterase gene, such as the aforementioned XYLD gene, into a plantwhich is being used in the methods of the invention.

[0052] Although, as described above, the method may be performed usingenzyme activities I, II and III which are provided by Ps. fluorescensbiovar. V, strain AN103 or mutants or variants thereof themselves, orintact-cell-free extracts thereof, it is preferred if at least one ofthe enzyme activities II and III is provided by a substantially purifiedenzyme. Substantially purified enzymes with enzyme activities II and IIIare described below.

[0053] In a particularly preferred embodiment of the invention themethod of the first aspect of the invention further comprises providinga compound, in addition to trans-ferulic acid or a salt thereof, whichmay be converted by any one of enzyme activities I, II or III into adesirable product. Suitably said compound is converted by any one ormore of said enzyme activities into a product which is found in, andpreferably contributes to the taste or aroma of, vanilla as extractedfrom Vanilla pod.

[0054] Vanilla as extracted from Vanilla pod contains vanillin as amajor component but also smaller quantities of desirable components suchas p-hydroxybenzoic acid, p-hydroxybenzaldehyde and vanillic acid.Typically these components, and vanillin, are present as glucosides ingreen vanilla pods as well as in the free form. However, upon hydrolysisor fermentation of the green pods or of hydrolysis of the fermentedpods, most of the components are present in the free form.

[0055] Thus, it is particularly preferred if said compound is any one oftrans-4-coumaric acid or a salt thereof, trans-4-coumaroyl SCoA,trans-caffeic acid or a salt thereof, trans-caffeoyl SCoA, or3,4-methylenedioxy-trans-cinnamic acid or a salt thereof. By the actionof one or more of enzyme activities I, II or III trans4-coumaric acid ora salt thereof and trans-4-coumaroyl SCoA are converted top-hydroxybenzaldehyde; trans-caffeic acid or a salt thereof andtrans-caffeoyl SCoA are converted to 3,4-dihydroxybenzaldehyde; and3,4methylenedioxy-trans-cinnamic acid or a salt thereof is converted toheliotropin.

[0056] It is preferred if the compound is trans4-coumaric acid or a saltthereof or trans-4-coumaroyl SCoA and that the desirable product is4-hydroxybenzaldehyde which is a significant component of naturalVanilla extract.

[0057] The enzyme activities I, II and III from Ps. fluorescens biovarV, strain AN103 are able to use trans-caffeate and trans-4-coumarate,(and, as appropriate, the products of their reaction with enzymeactivity I) with reasonable efficiency whereas cinnamate and3,4-methylenedioxy-trans-cinnamate, although may be used as substrates,are poor substrates of the AN103 enzymes.

[0058] Thus, the method of the first aspect of the invention is suitedto make vanilla flavourings and aromas which more closely resemble thevanilla from Vanilla pod.

[0059] The method of the first aspect of the invention may, in certaincircumstances, also be performed using the host cells and geneticallymodified cells and organisms as described below in more detail.

[0060] A second aspect of the invention provides a method of producingvanillic acid, or a salt thereof, comprising the steps of

[0061] (1) providing trans-ferulic acid or a salt thereof;

[0062] (2) providing trans-ferulate:CoASH ligase activity,trans-feruloyl SCoA hydratase activity, and4-hydroxy-3-methoxyphenyl-β-hydroxy-propionyl SCoA (HMPHP SCoA) cleavageactivity; and

[0063] (3) providing an activity that interconverts vanillin andvanillic acid.

[0064] For convenience, the activity that interconverts vanillin andvanillic acid is called enzyme activity IV. Conveniently the activity isprovided by vanillin:NAD⁺ oxidoreductase (vanillin dehydrogenase).Suitably, this activity is provided by Ps. fluorescens biovar. V, strainAN103. Methods of converting vanillin to vanillic acid or a salt thereofare also known in the art, for example Perestelo et al (1989) App.Environ. Microbiol. 55, 1660-1662 describes the production of vanillicacid from vanillin by resting cells of Serratia marcescens and Pomelto &Crawford (1983) App. Environ. Microbiol. 45, 1582-1585 describewhole-cell bioconversion of vanillin to vanillic acid by Streptomycesviridosporus.

[0065] The method of producing vanillic acid provided by the inventiontherefore includes the steps of exposing trans-ferulic acid or a saltthereof to enzyme activity I and forming a product, exposing the saidproduct of enzyme activity I to enzyme activity II to form a product,exposing the said product of enzyme activity II to enzyme activity IIIto form a product, and exposing the said product of enzyme activity IIIto enzyme activity IV to form a product.

[0066] It will be appreciated that vanillic acid can be made by the samemeans as vanillin is made in the method of the first aspect of theinvention provided, of course, that enzyme activity IV is supplied.

[0067] A further preferred embodiment of the first aspect of theinvention comprises the further step of separating vanillin from theother reaction components.

[0068] Vanillin, and other aromatic aldehydes, are, for example,recoverable by extraction with solvent, including supercritical carbondioxide, and by organophilic pervaporation, using membranes constructedof hydrophobic polymers (G. Bengston and K. W. Bodekker, in “Bioflavour95”, eds. P. Étiévant and P. Schreier, INRA, Paris, pp 393-403 (1995);S. M. Zhang and E. Drioli, Separ. Sci. Technol. 8, 1-31 (1994));pervaporation technology has been applied, for example, to the recoveryof flavour compounds of wine (N. Rajagopalan and M. Cheryan, J. MembraneSci. 104, 243-250 (1995)). Solid-phase extraction, followed bydesorption with solvent, is also possible, though less preferred.

[0069] However, in some circumstances, particularly where minor reactionproducts are present which are similar to compounds present in thevanilla isolated from Vanilla pod, vanillin is not isolated.

[0070] A further preferred embodiment of the second aspect of theinvention comprises the further step of separating vanillic acid or asalt thereof from the other reaction components.

[0071] Vanillic acid and other carboxylic acids may, for example, berecovered by solid-phase extraction, by solvent extraction under acidicconditions, or by pertraction; for example, L. Boyadzhiev and I.Atanassova (Process Biochemistry 29, 237-243 (1994)) describe therecovery of the aromatic amino acid, phenylalanine, by pertraction.

[0072] A third aspect of the invention provides Pseudomonas fluorescensbiovar. V, strain AN103 as deposited under the Budapest Treaty at theNational Collections of Industrial and Marine Bacteria Limited, AURISBusiness Centre, 23 St Machar Drive, Aberdeen AB2 1RY, Scotland underAccession No NCIMB 40783, or a mutant or variant thereof. Preferredmutants and variants are the same as those preferred in the first aspectof the invention. A particularly preferred mutant of Ps. fluorescensbiovar. V, strain AN103 is one which accumulates vanillin when providedwith trans-ferulic acid or a salt thereof. Conveniently, this is amutant of Ps. fluorescens biovar. V, strain AN103 wherein vanillin:NAD⁺oxidoreductase activity is absent or reduced. Suitably, there is amutation in the gene encoding vanillin:NAD⁺ oxidoreductase such that theenzyme activity is absent or substantially reduced. Such a mutant can bemade as described above.

[0073] A fourth aspect of the invention provides a polypeptide which, inthe presence of appropriate cofactors if any, is capable of catalysingthe interconversion of trans-feruloyl SCoA and4-hydroxy-3-methoxy-phenyl-β-hydroxypropionyl SCoA (HMPHP SCoA). Such apolypeptide has enzyme activity II. Conveniently, the polypeptidecomprises trans-feruloyl SCoA hydratase; more conveniently thepolypeptide comprises trans-feruloyl SCoA hydratase from Ps. fluorescensbiovar. V, strain AN103 or fragments or variants thereof which have atleast 1% of the specific activity of the native enzyme (in relation totrans-feruloyl SCoA hydratase activity), preferably at least 10%, morepreferably at least 100%.

[0074] The enzyme activity is readily purified as described in theExamples. Modifications to this procedure may be readily made by theperson skilled in the art so that a polypeptide with enzyme activity IIcan be obtained from any suitable source making use of the enzymeactivity II assay procedure described in the Examples.

[0075] It is preferred if the polypeptide of the fourth aspect of theinvention comprises the amino acid sequence.MetSerThrTyrGluGlyArgTrpLysThrValLysValGluIleGluAspGlyIleAlaPheValIleLeuAsnArgProGluLysArgAsnAlaMetSerProThrLeuAsnArgGluMetIleAspValLeuGluThrLeuGluGlnAspProAlaAlaGlyValLeuValLeuThrGlyAlaGlyGluAlaTrpThrAlaGlyMetAspLeuLysGluTyrPheArgGluValASpAlaGlyProGluIleLeuGlnGluLysIleArgArgGluAlaSerGlnTrpGlnTrpLysLeuLeuArgMetTyrAlaLysProThrlleAlaMetValAsnGlyTrpCysPheGlyGlyGlyPheSerProLeuValAlaGysAspLeuAlaIleCysAlaAspGluAlaThrPheGlyLeuSerGluIleAsnTrpGlyIleProProGlyAsnLeuValSarLysAlaMetAlaAspThrValGlyHisArgGlnSerLeuTyrTyrIleMetThrGlyLysThrPheGlyGlyGlnLysAlaAlaGluMetGlyLeuValAsnGluSerValProLeuAlaGlnLeuArgGluValThrIleGluLeuAlaArgAsnLeuLeuGluLysAsnProValValLeuArgAlaAlaLysHisGlyPheLysArgCysArgGluLeuThrTrpGluGlnAsnGluAspTyrLeuTyrAlaLysLeuAspGlnSerArgLeuLeuAspThrGluGlyGlyArgGluGlnGlyMetLysGlnPheLeuAspAspLysSerIleLysProGlyLeuGlnAlaTyrLysArg (SEQ ID No 2),

[0076] or a fragment or variant thereof.

[0077] The amino acid sequence is that given in FIG. 12 as that encodedby nucleotides 2872 to 3699.

[0078] A fifth aspect of the invention provides a polypeptide which, inthe presence of appropriate cofactors if any, is capable of catalysingthe interconversion of 4-hydroxy-3-methoxyphenyl-β-hydroxy-propionylSCoA (HMPHP SCoA) and vanillin. Such a polypeptide has enzyme activityIII. Conveniently, the polypeptide comprises HMPHP SCoA cleavage enzyme;more conveniently the polypeptide comprises HMPHP SCoA cleavage enzymefrom Ps. fluorescens biovar. V, strain AN103 or fragments or variantsthereof which have at least 1% of the specific activity of the nativeenzyme (in relation to HMPHP SCoA cleavage activity), preferably atleast 10%, more preferably at least 100%.

[0079] The enzyme activity is readily purified as described in theExamples. Modifications to this procedure may be readily made by theperson skilled in the art so that a polypeptide with enzyme activity IIIcan be obtained from any suitable source-making use of the enzymeactivity III assay procedure described in the Examples.

[0080] It is preferred if the polypeptide of the fifth aspect of theinvention comprises the amino acid sequenceMetSerThrTyrGluGlyArgTrpLysThrValLysValGluIleGluAspGlyIleAlaPheValIleLeuAsnArgProGluLysArgAsnAlaMetSerProThrLeuAsnArgGluMetIleAspValLeuGluThrLeuGluGlnAspProAlaAlaGlyValLeuValLeuThrGlyAlaGlyGluAlaTrpThrAlaGlyMetAspLeuLysGluTyrPheArgGluValAspAlaGlyProGluIleLeuGlnGluLysIleArgArgGluAlaSerGlnTrpGlnTrpLysLeuLeuArgMetTyrAlaLysProThrIleAlaMetValAsnGlyTrpCysPheGlyGlyGlyPheSerProLeuValAlaCysAspLeuAlaIleCysAlaAspGluAlaThrPheGlyLeuSerGluIleAsnTrpGlyIleProProGlyAsnLeuValSerLysAlaMetAlaAspThrValGlyHisArgGlnSerLeuTyrTyrIleMetThrGlyLysThrPheGlyGlyGlnLysAlaAlaGluMetGlyLeuValAsnGluSerValProLeuAlaGlnLeuArgGluValThrIleGluLeuAlaArgAsnLeuLeuGluLysAsnProValValLeuArgAlaAlaLysHisGlyPheLysArgCysArgGluLeuThrTrpGluGlnAsnGluAspTyrLeuTyrAlaLysLeuAspGlnSerArgLeuLeuAspThrGluGlyGlyArgGluGlnGlyMetLysGlnPheLeuAspAspLysSerIleLysProGlyLeuGlnAlaTyrLysArg (SEQ ID No 2),

[0081] or a fragment or variant thereof.

[0082] The amino acid sequence is that given in FIG. 12 as that encodedby nucleotides 2872 to 3699.

[0083] A sixth aspect of the invention provides a polypeptide comprisingthe amino acid sequenceMetLeuAspValProLeuLeuIleGlyGlyGlnSerCysProAlaArgAspGlyArgThrPheGluArgArgAsnProValThrGlyGluLeuValSerArgValAlaAlaAlaThrLeuGluAspA1lAspAlaAlaValAlaAlaAlaGlnGlnAlaPheProAlaTrpAlaAlaLeuAlaProAsnGluArgArgSerArgLeuLeuLysAlaAlaGluGlnLeuGlnAlaArgSerGlyGluPheIleGluAlaAlaGlyGluThrGlyAlaMetAlaAsnTrpTyrGlyPheAsnValArgLeuAlaAlaAsnMetLeuArgGluAlaAlaSerMetThrThrGlnValAsnGlyGluValIleProSerAspValProGlySerPheAlaMetAlaLeuArgGlnProCysGlyValValLeuGlyIleAlaProTrpAsnAlaProValIleLeuAlaThrArgAlaIleAlaMetProLeuAlaCysGlyAsnThrValValLeuLysAlaSerGluLeuSerProAlaValHisArgLeuIleGlyGlnValLeuGlnAspAlaGlyLeuGlyAspGlyValValAsnValIleSerAsnAlaProAlaAspAlaAlaGlnIleValGluArgLeuIleAlaAsnProAlaValArgArgValAsnPheThrGlySerThrHisValGlyArgIleValGlyGluLeuSerAlaArgHisLeuLysProAlaLeuLeuGluLeuGlyGlyLysAlaProLeuLeuValLeuAspAspAlaAspLeuGluAlaAlaValGlnAlaAlaAlaPheGlyAlaTyrPheAsnGlnGlyGlnIleCysMetSerThrGluArgLeuIleValAspAlaLysValAlaAspAlaPheValAlaGlnLeuAlaAlaLysValGluThrLeuArgAlaGlyAspProAlaAspProGluSerValLeuGlySerLeuValAspAlaSerAlaGlyThrArgIleLysAlaLeuIleAspAspAlaValAlaLysGlyAlaArgLeuValIleGlyGlyGlnLeuGluGlySerIleLeuGlnProThrLeuLeuAspGlyValAspAlaSerMetArgLeuTyrArgGluGluSerPheGlyProValAlaValValLeuArgGlyGluGlyGluGluAlaLeuLeuGlnLeuAlaAsnAspSerGluPheGlyLeuSerAlaAlaIlePheSerArgAspThrGlyArgAlaLeuAlaLeuAlaGlnArgValGluSerGlyIleCysHisIleAsnGlyProThrValHisAspGluAlaGlnMetProPheGlyGlyValLysSerSerGlyTyrGlySerPheGlyGlyLysAlaSerIleGluHisPheThrGlnLeuArgTrpValThrLeuGlnAsnGlyProArgHisTyr ProIle

[0084] (SEQ ID No 4) or a fragment or variant thereof.

[0085] The amino acid sequence is that given in FIG. 12 as that encodedby nucleotides 3804 to 5249.

[0086] As described in detail in the examples, this polypeptide sequenceencodes an enzyme with vanillin:NAD⁺ oxidoreductase activity from Ps.fluorescens biovar V., strain AN103.

[0087] By “variants” we include deletions, insertions and substitutionseither conservative or non-conservative, where such changes may reduceor enhance the activity, or may not substantially alter the activity. Inparticular, the seventh aspect of the invention includes the completepolypeptide sequence of Ps. fluorescens biovar V, strain AN103vanillin:NAD⁺ oxidoreductase and this polypeptide itself.

[0088] By “conservative substitutions” is intended combinations such asGly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; andPhe, Tyr. Such variants may be made using the methods of proteinengineering and site-directed mutagenesis as described below and as iswell known in the art.

[0089] A preferred embodiment of the invention is a polypeptide asdefined in the fourth, fifth or sixth of the invention which issubstantially pure.

[0090] By “substantially pure” we mean that the polypeptide issubstantially free of other polypeptides, or other macromolecules, withwhich it is usually found in nature. Suitably, the polypeptide issubstantially free of any other polypeptides or macromolecules. It ispreferred if the polypeptide has less than 50% by weight of any otherpolypeptide, preferably less than 10%, more preferably less than 1%,still more preferably less than 0.1% and most preferably less than0.01%.

[0091] Polypeptides can be purified using methods known in the art. Itis preferred if the polypeptide is the product of a recombinant DNA.

[0092] A single polypeptide chain may comprise more than one of theenzyme activities (II) trans-feruloyl SCoA hydratase activity or (III)HMPHP SCoA cleavage activity.

[0093] Our data in the Examples shows that, in the case of Ps.fluorescens biovar. V, strain AN103, enzyme activities II and III arefound in the same polypeptide chain, the sequence of which is given asthe preferred polypeptides of the fifth and sixth aspects of theinvention. Thus, when enzyme activities II and III are provided in anyaspect of the invention it is most convenient if they are provided inthe same polypeptide chain.

[0094] It will be appreciated that, using protein engineering methods orchemical cross-linking it may be possible to produce a single moleculewhich has enzyme activities II and III. Such a molecule, therefore,forms a further aspect of the invention.

[0095] An seventh aspect of the invention provides a polynucleotideencoding a polypeptide as defined in any one of the fourth, fifth orsixth aspects of the invention.

[0096] By “polynucleotide” we include RNA and DNA. DNA is preferred.

[0097] Thus, this aspect of the invention provides a polynucleotidewhich encodes any one of a polypeptide which has (II) trans-feruloylSCoA hydratase activity; (III) HMPHP SCoA cleavage activity; (IV)vanillin:NAD⁺ oxidoreductase activity; or a polypeptide which has morethan one of these activities. Preferably the polynucleotide is derivedfrom Ps. fluorescens biovar. V, strain AN103. A preferred polynucleotidecomprises all or at least a part of the Ps. fluorescens DNA containedwithin the cosmid clone pFI793 as deposited under the Budapest Treaty atthe National Collections of Industrial and Marine Bacteria Limited,AURIS Business Centre, 23 St Machar Drive, Aberdeen AB2 1RY, Scotlandunder Accession No NCIMB 40777, or a fragment or variant thereof.

[0098] The isolation of the cosmid clone pFI 793 is described in Example5; pFI 793 includes DNA which encodes polypeptides which have enzymeactivities II, III and IV. The cosmid clone pFI 793 itself, the genescontained in the Ps. fluorescens DNA thereof, and variants thereof formseparate aspects of the invention.

[0099] A variant of a polynucleotide includes any insertion, deletion orsubstitution of the sequence which encodes a fragment or variant of apolypeptide as defined above.

[0100] For example, site-directed mutagenesis or other techniques can beemployed to create single or multiple mutations, such as replacements,insertions, deletions, and transpositions, as described in Botstein andShortle, Strategies and Applications of In Vitro Mutagenesis, Science,229: 193-210 (1985), which is incorporated herein by reference. Sincesuch modified polynucleotides can be obtained by the application ofknown techniques to the teachings contained herein, such modifiedpolynucleotides are within the scope of the claimed invention.

[0101] Moreover, it will be recognised by those skilled in the art thatthe polynucleotide sequence (or fragments thereof) of the invention canbe used to obtain other DNA sequences that hybridise with it underconditions of high stringency. Such DNA includes any genomic DNA.

[0102] Accordingly, the polynucleotide of the invention includes DNAthat shows at least 55 percent, preferably 60 percent, and mostpreferably 70 percent homology with the polynucleotide sequencesidentified in the invention, provided that such homologous DNA encodes aprotein which is usable in the methods described herein.

[0103] DNA-DNA, DNA-RNA and RNA-RNA hybridisation may be, performed inaqueous solution containing between 0.1XSSC and 6XSSC and attemperatures of between 55° C. and 70° C. It is well known in the artthat the higher the temperature or the lower the SSC concentration themore stringent the hybridisation conditions. By high stringency we mean2XSSC and 65° C. 1XSSC is 0.15M NaCl/0.015M sodium citrate.

[0104] “Variants” of the polynucleotide include polynucleotides in whichrelatively short stretches (for example 20 to 50 nucleotides) have ahigh degree of homology (at least 50% and preferably at least 90 or 95%)with equivalent stretches of the polynucleotide of the invention eventhough the overall homology between the two polynucleotides may be muchless. This is because important active or binding sites may be sharedeven when the general architecture of the protein is different.

[0105] A particularly preferred polynucleotide comprises the nucleotidesequence ATGAGCACATACGAAGGTCGCTGGAAAACGGTCAAGGTCGAAATCGAAGACGGCATCGCGTTTGTCATCCTCAATCGCCCGGAAAAACGCAACGCGATGAGCCCGACCCTGAACCGCGAGATGATCGATGTTCTGGAAACCCTCGAGCAGGACCCTGCCGCCGGTGTGCTGGTGCTGACCGGTGCGGGCGAAGCCTGGACCGCAGGCATGGACCTCAAGGAATACTTCCGCGAAGTGGACGCCGGCCCGGAAATCCTCCAGGAAAAAATCCGCCGCGAAGCCTCGCAATGGCAATGGAAACTGCTGCGCATGTACGCCAAGCCGACCATCGCCATGGTCAATGGCTGGTGCTTCGGCGGCGCTTTCAGCCCGCTGGTGGCCTGCGACCTGGCGATCTGCGCCGACGAAGCAACCTTCGGTCTCTCGGAAATCAACTGGGGTATCCCCCCGGGCAACCTGGTGAGCAAGGCCATGGCCGACACCGTGGGCCACCGCCAGTCGCTCTACTACATCATGACCGGCAAGACCTTCGGTGGGCAGAAAGCCGCCGAGATGGGCCTGGTCAACGAAAGCGTGCCCCTGGCGCAACTGCGCGAAGTCACCATCGAGCTGGCGCGTAACCTGCTCGAAAAAAACCCGGTGGTGCTGCGTGCCGCCAAACACGGTTTCAAACGCTGCCGCGAACTGACCTGGGAGCAGAACGAGGATTACCTGTACGCCAAGCTCGATCAGTCGCGTTTGCTGGACACCGAAGGCGGTCGCGAGCAGGGCATGAAGCAATTCCTCGACGACAAGAGCATCAAGCCTGGCCTGCAAGCGTAAACGC (SEQ ID No 1)

[0106] (as given in FIG. 12, nucleotides 2872 to 5249) or a fragment orvariant thereof.

[0107] This polynucleotide encodes HMPHP SCoA cleavage enzyme activityand a trans-feruloyl SCoA hydratase activity from Ps. fluorescensbiovar. V, strain AN103 and encodes the preferred polypeptide of thefifth and sixth aspects of the invention.

[0108] A further particularly preferred polynucleotide comprises thenucleotide sequenceATGCTGGACGTGCCCCTGCTGATTGGCGGCCAGTCGTGCCCCGCGGCGCGACGGTCGAACCTTCGAGCGCCGCAACCCGGTGACTGGCGAGTTGGTGTCGCGGGTTGCCGCCGCCACCCTGGAAGATGCCGACGCCGCCGTGGCCGCTGCCCAGCAAGCGTTTCCCGCGTGGGCCGCGCTGGCGCCCAATGAACGGCGCAGCCGTTTGCTCAAGGCCGCCGAACAATTGCAGGCGCGCAGCGGCGAGTTCATCGAGGCGGCGGGCGAGACCGGCGCCATGGCCAACTGGTACGGCTTCAACGTACGGCTGGCGGCCAACATGCTGCGTGAAGCGGCATCGATGACCACCCAGGTCAATGGTGAAGTGATTCCCTCGGACGTTCCCGGCAGTTTCGCCATGGCCCTGCGCCAGCCCTGTGGCGTGGTGCTGGGCATCGCCCCCTGGAACGCCCCGGTGATTCTCGCCACCCGGGCGATTGCCATGCCGCTGGCCTGTGGCAACACCGTGGTGCTGAAGGCTCCGAGCTGAGTCCGGCGGTGCATCGCTTGATCGGCCAGGTGCTGCAGGACGCCGGCCTGGGCGATGGCGTGGTCAACGTCATCAGTAATGCGCCGGCGGATGCGGCACAGATTGTCGAGCGCCTGATTGCCAACCCGGCCGTACGCCGGGTCAATTTCACCGGTTCGACCCACGTCGGGCGCATTGTCGGCGAGCTCTCGGCGCGCCACCTCAAACCGGCGTTGCTCGAGCTGGGCGGCAAGGCACCGTTGCTGGTGCTCGATGATGCCGACCTGGAGGCTGCCGTGCAGGCGGCGGCGTTTGGCGCCTACTTCAACCAGGGACAGATCTGTATGTCCACCGAGCGCCTGATTGTCGATGCCAAGGTGGCCGACGCCTTTGTCGCCCAGTTGGCGGCCAAGGTCGAGACCCTGCGCGCCGGTGATCCTGCCGACCCGGAGTCGGTGCTCGGTTCGCTGGTGGACGCCAGCGCTGGCACGCGGATCAAAGCGTTGATCGATGATGCCGTGGCCAAGGGCGCGCGCCTGGTAATCGGCGGGCAACTGGAGGGCAGCATCTTGCAGCCGACCCTGCTCGACGGTGTCGACGCGAGCATGCGTTTGTACCGCGAAGAGTCCTTCGGCCCGGTGGCGGTGGTGCTGCGCGGCGAGGGCGAAGAAGCGCTGTTGCAACTGGCCAACGACTCCGAGTTCGGTTTGTCGGCGGCGATTTTCAGTCGTGACACCGGCCGTGCCCTGGCCCTGGCCCAGCGGGTCGAATCGGGCATCTGCCACATCAACGGCCCGACCGTGCACGACGAAGCGCAAATGCCTTTTGGCGGGGTCAAGTCCAGCGGCTACGGCAGTTTTGGCGGCAAGGCATCGATTGAGCATTTCACTCAGTTGCGCTGGGTCACCCTCCAGAATGGTCCACGGCACTAT CCGATC

[0109] (SEQ ID No 3) (as given in FIG. 12, nucleotides 3804 to 5249)

[0110] or a fragment or variant thereof. This polynucleotide encodes thepolypeptide sequence of the sixth aspect of the invention. It isparticularly convenient to isolate the whole gene from cosmid clone pFI793 as deposited under the Budapest Treaty at NCIMB under Accession NoNCIMB 40777.

[0111] The polynucleotides of the invention are all readily isolatedfrom pFI 793 by probing with the given sequences or parts thereof, or byother methods known in the art, and the nucleotide sequences can beconfirmed by reference to the deposited cosmid (pFI 793).

[0112] It will be appreciated that fragments and variants of thepolynucleotides of the invention can readily be made by the personskilled in the art using standard molecular biological methods such asthose described in Sambrook et al “Molecular Cloning, a laboratorymanual”, (1989), (2nd Edition), Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. The whole gene and variants and fragmentsthereof are specifically included in this aspect of the invention.

[0113] It is preferred if the polynucleotide, conveniently DNA, isjoined to a nucleic acid vector.

[0114] DNA constructs of the invention may be purified from the hostcell using well known methods.

[0115] For example, plasmid vector DNA can be prepared on a large scalefrom cleaved lysates by banding in a CsCl gradient according to themethods of Clewell & Helinski (1970) Biochemistry 9, 4428-4440 andClewell (1972) J. Bacteriol. 110, 667-676. Plasmid DNA extracted in thisway can be freed from CsCl by dialysis against sterile, pyrogen-freebuffer through Visking tubing or by size-exclusion chromatography.

[0116] Alternatively, plasmid DNA may be purified from cleared lysatesusing ion-exchange chromatography, for example those supplied by Qiagen(Chatsworth, Calif., USA). Hydroxyapatite column chromatography may alsobe used.

[0117] The DNA is then expressed in a suitable host to produce apolypeptide of the invention. Thus, a DNA encoding a polypeptide of theinvention may be used in accordance with known techniques, appropriatelymodified in view of the teachings contained herein, to construct anexpression vector, which is then used to transform an appropriate hostcell for the expression sand production of the polypeptide of theinvention. Such techniques include those disclosed in U.S. Pat. Nos.4,440,859 issued Apr. 3, 1984 to Rutter et al, 4,530,901 issued Jul. 23,1985 to Weissman, 4,582,800 issued Apr. 15, 1986 to Crowl, 4,677,063issued Jun. 30, 1987 to Mark et al, 4,678,751 issued Jul. 7, 1987 toGoeddel, 4,704,362 issued Nov. 3, 1987 to Itakura et al, 4,710,463issued Dec. 1, 1987 to Murray, 4,757,006 issued Jul. 12, 1988 to Toole,Jr. et al, 4,766,075 issued Aug. 23, 1988 to Goeddel et al, and4,810,648 issued Mar. 7, 1989 to Stalker, all of which are incorporatedherein by reference.

[0118] The DNA encoding the polypeptide constituting the compound of theinvention may be joined to a wide variety of other DNA sequences forintroduction into an appropriate host. The companion DNA will dependupon the nature of the host, the manner of the introduction of the DNAinto the host, and whether episomal maintenance or integration isdesired.

[0119] Generally, the DNA is inserted into an expression vector, such asa plasmid, in proper orientation and correct reading frame forexpression. If necessary, the DNA may be linked to the appropriatetranscriptional and translational regulatory control nucleotidesequences recognised by the desired host, although such controls aregenerally available in the expression vector. The vector is thenintroduced into the host through standard techniques. Generally, not allof the hosts will be transformed by the vector. Therefore, it will benecessary to select for transformed host cells. One selection techniqueinvolves incorporating into the expression vector a DNA sequence, withany necessary control elements, that codes for a selectable trait in thetransformed cell, such as antibiotic resistance. Alternatively, the genefor such selectable trait can be on another vector, which is used toco-transform the desired host cell.

[0120] Host cells that have been transformed by the recombinant DNA ofthe invention may then be cultured for a sufficient time and underappropriate conditions known to those skilled in the art in view of theteachings disclosed herein to permit the expression of the polypeptide,which, if desirable, can then be recovered.

[0121] Many expression systems are known, including bacteria (forexample Escherichia coli and Bacillus subtilis), yeasts (for exampleSaccharomyces cerevisiae), filamentous fungi (for example Aspergillus),plant cells and whole plants, animal cells and insect cells.

[0122] The vectors include a prokaryotic replicon, such as the Co1E1ori, for propagation in a prokaryote, even if the vector is to be usedfor expression in other, non-prokaryotic, cell types. The vectors canalso include an appropriate promoter such as a prokaryotic promotercapable of directing the expression (transcription and translation) ofthe genes in a bacterial host cell, such as E. coli transformedtherewith.

[0123] Several promoters are available to direct transcription ofbacterial and other heterologous genes in plants. In particular, theseinclude the 35S promoter of cauliflower mosaic virus (CaMV 35S), theribulose bisphosphate carboxylase small subunit promoter and theAgrobacterium T-DNA octopine synthase and manopine synthase promoters.These promoters have been widely used, for example, in conjunction withbacterial genes conferring herbicide resistance (see D. M. Stalker,ibid., pp 82-104). These promoters do not confer any specificity of geneexpression at the organ, tissue or organellar levels, or responsivenessof gene expression to environmental influences such as light.

[0124] A promoter is an expression control element formed by a DNAsequence that permits binding of RNA polymerase and transcription tooccur. Promoter sequences compatible with exemplary bacterial hosts aretypically provided in plasmid vectors containing convenient restrictionsites for insertion of a DNA segment of the present invention.

[0125] Typical prokaryotic vector plasmids are pUC18, pUC19, pBR322 andpBR329 available from Biorad Laboratories, (Richmond, Calif., USA) andpTrc99A and pKK223-3 available from Pharmacia, Piscataway, N.J., USA.

[0126] A typical mammalian cell vector plasmid is pSVL available fromPharmacia, Piscataway, N.J., USA. This vector uses the SV40 latepromoter to drive expression of cloned genes, the highest level ofexpression being found in T antigen-producing cells, such as COS-1cells.

[0127] An example of an inducible mammalian expression vector is pMSG,also available from Pharmacia. This vector uses theglucocorticoid-inducible promoter of the mouse mammary tumour virus longterminal repeat to drive expression of the cloned gene.

[0128] Useful yeast plasmid vectors are pRS403-406 and pRS413-416 andare generally available from Stratagene Cloning Systems, La Jolla,Calif. 92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are YeastIntegrating plasmids (YIps) and incorporate the yeast selectable markersHIS3, TRP1, LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromereplasmids (YCps)

[0129] A variety of methods have been developed to operably link DNA tovectors via complementary cohesive termini. For instance, complementaryhomopolymer tracts can be added to the DNA segment to be inserted to thevector DNA. The vector and DNA segment are then joined by hydrogenbonding between the complementary homopolymeric tails to formrecombinant DNA molecules.

[0130] Synthetic linkers containing one or more restriction sitesprovide an alternative method of joining the DNA segment to vectors. TheDNA segment, generated by endonuclease restriction digestion asdescribed earlier, is treated with bacteriophage T4 DNA polymerase or E.coli DNA polymerase I, enzymes that remove protruding,3′-single-stranded termini with their 3′-5′-exonucleolytic activities,and fill in recessed 3′-ends with their polymerizing activities.

[0131] The combination of these activities therefore generatesblunt-ended DNA segments. The blunt-ended segments are then incubatedwith a large molar excess of linker molecules in the presence of anenzyme that is able to catalyze the ligation of blunt-ended DNAmolecules, such as bacteriophage T4 DNA ligase. Thus, the products ofthe reaction are DNA segments carrying polymeric linker sequences attheir ends. These DNA segments are then cleaved with the appropriaterestriction enzyme and ligated to an expression vector that has beencleaved with an enzyme that produces termini compatible with those ofthe DNA segment.

[0132] Synthetic linkers containing a variety of restrictionendonuclease sites are commercially available from a number of sourcesincluding International Biotechnologies Inc, New Haven, Conn., USA.

[0133] A desirable way to modify the DNA encoding the polypeptide of theinvention is to use the polymerase chain reaction as disclosed by Saikiet al (1988) Science 239, 487-491.

[0134] In this method the DNA to be enzymatically amplified is flankedby two specific oligonucleotide primers which themselves becomeincorporated into the amplified DNA. The said specific primers maycontain restriction endonuclease recognition sites which can be used forcloning into expression vectors using methods known in the art.

[0135] The present invention also relates to a host cell transformedwith a polynucleotide vector construct of the present invention. Thehost cell can be either prokaryotic or eukaryotic and it may becomprised in a multicellular organism such as a plant. Bacterial cellsare preferred prokaryotic host cells and typically are a strain of E.coli such as, for example, the E. coli strains DH5 available fromBethesda Research Laboratories Inc., Bethesda, Md., USA, and RR1available from the American Type Culture Collection (ATCC) of Rockville,Md., USA (No ATCC 31343). Preferred eukaryotic host cells include yeastand plant cells. Yeast host cells include YPH499, YPH500 and YPH501which are generally available from Stratagene Cloning Systems, La Jolla,Calif. 92037, USA. Preferred plant host cells and plants include thosefrom Nicotiana spp., Solanum tuberosum (potato), Brassica spp. (eg oilseed rape), Beta spp. (eg sugar beet, leaf beet and beetroot), Capsicumspp. and Vanilla spp.

[0136] Transformation of appropriate cell hosts with a DNA construct ofthe present invention is accomplished by well known methods thattypically depend on the type of vector used. With regard totransformation of prokaryotic host cells, see, for example, Cohen et al(1972) Proc. Natl. Acad. Sci. USA 69, 2110 and Sambrook et al (1989)Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. Transformation of yeast cells is described inSherman et al (1986) Methods In Yeast Genetics, A Laboratory Manual,Cold Spring Harbor, N.Y. The method of Beggs (1978) Nature 275, 104-109is also useful. With regard to plant cells and whole plants three planttransformation approaches are typically used (J. Draper and R. Scott inD. Grierson (ed.), “Plant Genetic Engineering”, Blackie, Glasgow andLondon, 1991, vol. 1, pp 38-81):

[0137] i) Agrobacterium-mediated transformation, using the Ti plasmid ofA. tumefaciens and the Ri plasmid of A. rhizogenes (P. Armitage, R.Walden and J. Draper in J. Draper, R. Scott, P. Armitage and R. Walden(eds.), “Plant Genetic Transformation and Expression—A LaboratoryManual”, Blackwell Scientific Publications, Oxford, 1988, pp 1-67; R. J.Draper, R. Scott and J. Hamill ibid., pp 69-160);

[0138] ii) DNA-mediated gene transfer, by polyethylene glycol-stimulatedDNA uptake into protoplasts, by electroporation, or by microinjection ofprotoplasts or plant cells (J. Draper, R. Scott, A. Kumar and G. Dury,ibid., pp 161-198);

[0139] iii) transformation using particle bombardment (D. McCabe and P.Christou, Plant Cell Tiss. Org. Cult., 3, 227-236 (1993); P. Christou,Plant J., 3, 275-281 (1992)).

[0140] Agrobacterium-mediated transformation is generally ineffectivefor monocotyledonous plants (eg Vanilla), for which approaches ii) andiii) are therefore preferred. In all approaches a suitable selectionmarker, such as kanamycin- or herbicide-resistance, is preferred oralternatively a screenable marker (“reporter”) gene, such asβ-glucuronidase or luciferase (see J. Draper and R. Scott in D. Grierson(ed.), “Plant Genetic Engineering”, Blackie, Glasgow and London, 1991,vol. 1 pp 38-81).

[0141] Electroporation is also useful for transforming cells and is wellknown in the art for transforming yeast cell, bacterial cells and plantcells.

[0142] For example, many bacterial species may be transformed by themethods described in Luchansky et al (1988) Mol. Microbiol. 2, 637-646incorporated herein by reference. The greatest number of transformantsis consistently recovered following electroporation of the DNA-cellmixture suspended in 2.5×PEB using 6250V per cm at 25 μFD.

[0143] Methods for transformation of yeast by electroporation aredisclosed in Becker & Guarente (1990) Methods Enzymol. 194, 182.

[0144] Successfully transformed cells, ie cells that contain a DNAconstruct of the present invention, can be identified by well knowntechniques. For example, cells resulting from the introduction of anexpression construct of the present invention can be grown to producethe polypeptide of the invention. Cells can be harvested and lysed andtheir DNA content examined for the presence of the DNA using a methodsuch as that described by Southern (1975) J. Mol. Biol. 98, 503 orBerent et al (1985) Biotech. 3, 208. Alternatively, the presence of theprotein in the supernatant can be detected using antibodies as describedbelow.

[0145] In addition to directly assaying for the presence of recombinantDNA, successful transformation can be confirmed by well knownimmunological methods when the recombinant DNA is capable of directingthe expression of the protein. For example, cells successfullytransformed with an expression vector produce proteins displayingappropriate antigenicity. Samples of cells suspected of beingtransformed are harvested and assayed for the protein using suitableantibodies.

[0146] Thus, in addition to the transformed host cells themselves, thepresent invention also contemplates a culture of those cells, preferablya monoclonal (clonally homogeneous) culture, or a culture derived from amonoclonal culture, in a nutrient medium; and also, in the case of plantcells, a plant derived from, and containing, such cells.

[0147] It is particularly preferred if the host cell comprises a nucleicacid which encodes any one of, or combination of, a polypeptide which,in the presence of appropriate cofactors if any is capable of catalysingthe interconversion of trans-feruloyl SCoA and4-hydroxy-3-methoxyphenyl-β-hydroxypropionyl SCoA (HMPHP SCoA) or apolypeptide which, in the presence of appropriate cofactors if any, iscapable of catalysing the interconversion of4-hydroxy-3-methoxyphenyl-β-hydroxypropionyl SCoA (HMPHP SCoA) andvanillin.

[0148] It will be appreciated that the host cells of the invention or anextract thereof are particularly suited for use in the methods of theinvention. It is particularly preferred if the host cell does notcontain means for converting vanillin to a non-vanillin product.

[0149] It is most preferred if the host cell is a plant cell or iscomprised in a whole plant or a bacterial cell or a yeast cell.Preferred bacterial hosts include lactic acid bacteria such asLactococcus spp. and Lactobacillus nspp. Preferred yeast hosts includeSaccharontyces cerevisiae and its biovars. It is particularly preferredif the host cell is a food-grade host cell (for example a microorganismwhich is used or can be used in the food or beverage industry). It isalso preferred if the plant is an edible plant.

[0150] It will be appreciated that some host cells or host organisms mayalready contain enzyme activities I, II, III or IV and, in that case, itmay be sufficient, in order to use the host cells in the methods of theinvention, to introduce into said host cell or host organism one or morepolynucleotides which encode enzyme activities II, III or IV whichencode those enzyme activities which are deficient in the host cell orhost organisms.

[0151] In the case of plants for use in the methods of the invention, itis preferred that the relevant gene expression is directed to targetorgans, tissues and subcellular organelles where trans-feruloyl SCoA, orother appropriate substrates (eg 4-trans-coumaroyl SCoA ortrans-caffeoyl ScoA) for the enzymes encoded by the transferred genes,are most readily available. The stage at which thioesterification withCoASH occurs in plants, in relation to the progressive substitution ofthe phenyl ring which takes place during the conversion fromtrans-cinnamate to trans-ferulate, is unclear and may be variable (seeR. Whetten and R. Sederoff, The Plant Cell, 7, 1001-1013 (1995)). Thesubcellular localisation or distribution of these intermediates duringplant phenylpropanoid metabolism also remains uncertain; it is likelythat they are cytosolic, or that some functional organisation of theenzymes which metabolise them occurs. The concept of the metaboliccluster or “metabolon”, in which there is a degree of metabolicchannelling and free diffusion is restricted, has been proposed anddiscussed (see R. A. Dixon and N. L. Paiva, The Plant Cell, 7, 1085-1097(1995) and Ioc cit.).

[0152] Trans-ferulate, 4-trans-coumarate and trans-caffeate are normalmetabolic intermediates. Thus, there may be no requirement to manipulatehost plants in order to provide trans-feruloyl SCoA. Theirconcentrations are expected to be influenced in varying degrees byphysiological requirements for a wide range of end-products of thephenylpropanoid pathway, including for example lignin, coumarins andflavonoids. There is some evidence that the activity of the first enzymeof the phenylpropanoid pathway, phenylalanine ammonia lyase (PAL: EC4.3.1.5), can influence the accumulation of end-products of the pathway(N. Bate, J. Orr, W. Ni, A. Meromi, T. Nadler-Hassar, P. W. Doerner, R.A. Dixon, C. J. Lamb and Y. Elkind, Proc. Natl. Acad. Sci. USA, 91,7608-7612 (1994)) so under certain circumstances it is possible toenhance the metabolic effects of the expression of the genes for enzymeactivities II, and III by increasing the expression of PAL.

[0153] It is well known that gene expression in the phenylpropanoidpathway is responsive to a range of environmental and stress factors,including wounding, chemical elicitors of pathogenic origin, and u/vlight. The mechanisms regulating these responses are not very wellunderstood, though several transcription factors have been identified(see R. A. Dixon and N. L. Paiva, The Plant Cell, 7, 1085-1097 (1995)).However, particularly when these are more fully characterised, they door will offer opportunities for predictable and inducible control ofgene expression, particular to enhance the provision of substrate.

[0154] Thus, a further aspect of the invention provides a transgenicplant comprising a polynucleotide according to any of the fourth orfifth aspects of the invention. In other words, the transgenic plant isgenetically engineered to encode and, preferably, express any one ormore of enzyme activities II or III. It is particularly preferred that,following said genetic engineering the plant is able to produce vanillinfrom trans-feruloyl SCoA. It will be appreciated that, depending on theenzymes present in the host plant, it may be necessary only to provide agene encoding only a single of said enzyme activities or it may benecessary to provide a gene or genes of any two of said enzymeactivities.

[0155] Conveniently, the transgenic plant is genetically engineered toencode and, preferably, express enzyme activities II and III. It can bereadily seen that the transgenic plants of this aspect of the inventionmay be used in the methods of producing vanillin of the invention,especially when the plant provides the enzyme activity thatinterconverts trans-ferulic acid or a salt thereof and trans-feruloylSCoA.

[0156] Preferably the plant is a plant which is readily transformed.Preferably the plant is a plant which is commonly used in agriculture orhorticulture and more preferably the plant is an edible plant.Advantageously the plant is a plant in which it is desirable tointroduce a vanilla flavour or aroma.

[0157] Preferred plants include those selected from Nicotiana spp.,Solanum tuberosum, Brassica spp., Capsicum spp., Beta spp. and Vanillaspp.

[0158] As is described in more detail below the plant may be eaten ormay be processed into a foodstuff or beverage. Thus, conveniently thetransgenic plant is processed or prepared so that it is not capable ofreproduction or cultivation, for example the transgenic plant isharvested from the environment in which it was grown.

[0159] When vanillin (or the desirable products such asp-hydroxybenzaldehyde) is produced in a host cell or organism of theinvention, especially if it is produced in a transgenic plant of theinvention, the vanillin or desirable product may initially be present inthe form of a glycoside, more particularly, a β-D-glycoside, or, in thecase of a carboxylic acid, as esters of β-D-glucose (as occurs inVanilla pod). In this case, it is desirable to release the vanillin (anddesirable product) into its uncombined form, for example by acid- orbase-catalysed hydrolysis or by the use of glycosides such as theβ-D-glucosidase (emulsin; S. Hestrin, D. S. Femgold and M. Schramm,Meth. Enzymol. I, 231-257 (1955); see also D. Chassagne, C. Bayonore, J.Crouzet and K. Baumes in “Bioflavour 95”, eds. P. Étiévant and P.Schreier, INRA, Paris, pages 217-222 (1995).).

[0160] In relation to the use of a microorganism such as Ps. fluorescensbiovar V, strain AN103 or of a microorganism which has been geneticallymodified to contain enzyme activities II and III (or at least those ofthese activities that it does not normally have), it is preferred thatsaid microorganism is provided with trans-feruloyl SCoA or a means toprovide said CoA thioester from trans-fernlic acid or a salt thereof atleast in its culture medium.

[0161] Thus, it can be seen from all of the foregoing description thatthe invention includes biochemical and fermentative processes forproducing vanillin and vanillic acid, recombinant or transgenic plantsand the use of the said plants in a method of making vanillin orvanillic acid.

[0162] Typically, in a biochemical process the strain of Pseudomonas (egPs. fluorescens biovar. V, strain AN 103) provides an enzyme system forthe biotransformation of plant derived trans-ferulic acid to vanillinand/or related compounds. Enzyme preparations, whole cells ofPseudomonas or a heterologous host organism expressing appropriatePseudomonas genes may be used for this. A variety of mutants ofPseudomonas and various additional enzyme preparations, co-factors orco-factor regenerating systems may be used. The Pseudomonas enzymesmight be overexpressed in a heterologous host before being extracted andused in a biotransformation.

[0163] Alternatively, but suitably, some form of fermentation processmay be used which involves the Pseudomonas strain or an appropriatederived mutant or a heterologous host organism in which the genes forbiotransformation are expressed. The chosen microorganism is typicallygrown on a ferulate-rich substrate or a substrate comprisingtrans-feruloyl SCoA. This could generate a vanillin production process.

[0164] In addition the invention includes recombinant microorganisms (eglactic acid bacteria) which are modified to contain genes encodingenzyme activities II and III. For example, lactic acid bacteria modifiedaccording to the invention may be used to produce vanilla-flavouredyoghurt provided that they are supplied with trans-feruloyl SCoA.

[0165] Advantageously, genes for vanillin production (such as thoseencoding enzyme activities II and III) are expressed in a variety ofplant species such that vanillin accumulates in an appropriate tissue.In this case a new crop plant may be cultivated and vanillin would beextracted. Thus, a sugar beet plant may be made according to theinvention in which the beet was rich in vanillin. In addition thedevelopment of a novel plant cultivars for direct consumption (egvanilla-flavoured capsicum), or even for their desirable aromaproperties, included in the invention.

[0166] The polypeptides of the invention, or the genes which encodethem, may be used either individually or in combination (whether assubstantially isolated polypeptides, or in cell-free extracts or as hostcells or organisms which encode and, preferably, express saidpolypeptides) to convert a compound into a desirable product. Certaincompounds and desirable products have been described above. However, theinvention also includes the production of any other desirable product,such as a flavour or aroma, from substrates related to trans-ferulateand other known substrates of the polypeptides (enzymes) of theinvention. Thus, the polypeptides or genes of the invention, eitherindividually or in combination, may be used in processes for converting,for example, synthetic substrates of the said polypeptides (enzymes)into novel flavours and aromas or they may be used to modify thechemical profile of known flavours or aromas.

[0167] Similarly, it will be appreciated that certain desirable productscan be made by the further action of enzyme activity IV upon certaincompounds, particularly those produced by enzyme activities I, II andIII. For example, enzymes activities I, II and III may be used toconvert trans-4-coumaric acid or trans4-coumaroyl SCoA top-hydroxybenzaldehyde and enzyme activity IV may then be used to convertp-hydroxybenzaldehyde to p-hydroxy-benzoic acid. Thus, the inventionincludes a method of producing p-hydroxy-benzoic acid using at least oneof enzyme activities I, II, III and IV and advantageously using all ofthem.

[0168] A further aspect of the invention provides a food or beveragecomprising a host cell comprising a polynucleotide of the invention, oran extract of said host cell. The host cells comprising one or morepolynucleotides of the invention, especially those such host cells whichproduce vanillin by virtue of the presence of said polynucleotide orpolynucleotides, may be used in the production of food or beverages. Inparticular, as discussed above, lactic acid bacteria which producevanillin by the methods of the invention may be used in the productionof cheese, yogurt and related products including milk drinks. Similarly,yeasts which produce vanillin by the methods of the invention may beused in the production of food and beverages such as bread and beer.Extracts of said host cells may also be used in the food or beverageindustry.

[0169] A still further aspect of the invention provides a food orbeverage comprising a transgenic plant comprising a polynucleotide ofthe invention, or a part or extract or said transgenic plant. Thetransgenic plant comprising one or more polynucleotides of theinvention, especially those such transgenic plants which producevanillin by virtue of the presence of said polynucleotide orpolynucleotides, may constitute the food itself or they may be processedto form the food or beverage. For example, a tuber of a transgenicpotato of the invention constitutes a food of this aspect of theinvention. Alternatively, said transgenic potato may be processed intoanother foodstuff which is, nevertheless, a food of this aspect of theinvention.

[0170] Still further aspects of the invention provide use of Pseudomonasfluorescens biovar. V, strain AN103 or a mutant or derivative thereof ina method for producing vanillin, or vanillic acid or salt thereof; useof a polypeptide of the invention in a method for producmg vanillin, orvanillic acid or salt thereof; use of a polynucleotide of the inventionin a method for producing vanillin, or vanillic acid or salt thereof;and use of a host cell of the invention in a method for producingvanillin or vanillic acid or a salt thereof.

[0171] As is clear from the foregoing the invention also includes amethod of producing vanillin or vanillic acid, or other relatedproducts, the method comprising providing trans-feruloyl SCoA (or anyother suitable CoASH thioester which can be acted upon by enzymeactivity II) and providing enzyme activity II, enzyme activity III and,in the case of vanillic acid production or another related product,enzyme activity IV. Trans-feruloyl SCoA is obtainable by the methoddescribed in Example 2 or it may be obtained using the methods of Zenket al (1980) Anal. Biochem. 101, 182-187, incorporated herein byreference. Other CoASH thioesters which may be substrates for enzyme IIare also described in Zenk et al.

[0172] The preferred method steps and organisms for use in the methods,and the foods and beverages of the earlier aspects of the invention arealso preferred in this method of the invention to the extent that theyare compatible with this method, and the organisms used in this method.

[0173] As is discussed above, trans-feruloyl SCoA (and related CoASHthioesters such as 4-trans-coumaroyl SCoA and trans-caffeoyl SCoA) arenormal metabolic intermediates in plants. Thus, a transgenic plant whichcomprises a polynucleotide or polynucleotides which encode, andpreferably express, enzyme activities II and III in a location in theplant which contains trans-feruloyl SCoA or other suitable CoASHthioester is particularly suited for the purposes of this aspect of theinvention. As is described above, such transgenic plants and productsderived therefrom form part of the invention.

[0174] The invention will now be described in more detail with referenceto the following Examples and Figures wherein:

[0175]FIG. 1 describes the vanillin pathway in Pseudomonas fluorescensbiovar. V, strain AN103. HMPHP SCoA is4-hydroxy-3-methoxy-phenyl-β-hydroxypropionyl SCoA. I is an enzyme thatcatalyses the interconversion of trans-ferulic acid and trans-feruloylSCoA; II is an enzyme that catalyses the interconversion oftrans-feruloyl SCoA and HMPHP SCoA; III is an enzyme that catalyses theinterconversion of HMPHP SCoA and vanillin; and IV is an enzyme thatcatalyses the interconversion of vanillin and vanillic acid.

[0176]FIG. 2 illustrates the growth of strain AN103 following transferto MM medium containing 10 mM vanillate (V), 10 mM trans-ferulate (F) or10 mM trans-ferulate plus 10 mM vanillate (FV). Cultures were previouslygrown in MM medium containing 10 mM vanillate.

[0177]FIG. 3 indicates the changes in trans-ferulate and vanillateconcentrations during growth of strain AN 103 on MM medium containing 10mM trans-ferulate.

[0178]FIG. 4 shows the production of vanillin (van) and vanillate (VA)by an extract of cells of strain AN103 (165 μg protein) incubated withtrans-ferulate, ATP, CoASH and Mg²⁺ ions, both in the absence of NAD⁺and in its presence (0.5 mM). Cells were grown in the presence of 10 mMtrans-ferulate, plus 10 mM vanillate.

[0179]FIG. 5 demonstrates the formation of feruloyl SCoA, vanillin andacetyl SCoA from trans-ferulate supplied to a PD10-treated extract oftrans-ferulate-grown cells of strain AN 103 (7kg protein) in thepresence of ATP, CoASH and Mg²⁺ ions.

[0180]FIG. 6 demonstrates the production of vanillin, acetyl SCoA andferuloyl SCoA from HMPHP SCoA supplied to a PD10-treated cell-freeextract (7 μg protein) of trans-ferulate-grown cells of strain AN103.

[0181]FIG. 7 shows the induction over time of trans-ferulate:CoASHligase activity in response to 10 mM trams-ferulate (F), 10 mM vanillate(V) and 10 mM trans-ferulate plus 10 mM vanillate (FV) present in MMmedium. The inocula were grown in MM medium plus 10 mM vanillate; growthconditions, enzyme extraction and assay were as described in Examples 1and 2.

[0182]FIG. 8 shows SDS-PAGE of A), an extract of cells grown in MMmedium with 10 mM trans-ferulate, electrophoresed at successive stagesof purification of the HMPHP SCoA cleavage enzyme; successive stages areCrude Extract, Mono Q-purified, Mono-P-purified and PhenylSuperose-purified, and B), extracts of cells grown in MM medium witheither 10 mM vanillate or 10 mM trans-ferulate and electrophoresedalongside Mono-P-purified cleavage enzyme; A) silver-stained; B)Coomassie-stained.

[0183]FIG. 9 shows EcoRI/PstI digests of cosmid clones pFI793, pFI794,pFI795 and pFI796 separated on an agarose gel.

[0184]FIG. 10 shows the sequence of the redundant primers designed from20 N-terminal amino residues of the 31-kDal protein (SEQ ID Nos. 5 and6).

[0185]FIG. 11 shows a Southern blot of EcoRI/PstI digests of variouscosmid clones probed with the PCR product amplified using the N-terminaldegenerate oligonucleotide primers as shown in FIG. 10.

[0186]FIG. 12 shows the nucleotide sequence of pFI989 (ie the 4370 bpEcoRI/Pstl fragment from pFI794), together with the succeeding 882 bpdetermined from a further subclone, pFI1056 and from pFI794 itself (SEQID No 7). The amino acid sequence of the 31 kD protein and thatcorresponding to the succeeding open reading frame encodingvanillin:NAD⁺ oxidoreductase (vanillin dehydrogenase) (SEQ ID Nos. 2 and4) are also shown.

[0187]FIG. 13 shows the nucleotide sequence of pFI901 (ie the 1.8 kbEcoRI/PstI fragment from pFI793) (SEQ ID No 8).

[0188]FIG. 14 shows the nucleotide sequence of pFI911 (ie the 850 bpEcoRI/PstI fragment from pFI793) (SEQ ID No 9).

[0189]FIG. 15 shows the nucleotide sequence of pFI912 (ie the 958 bpEcoRI/PstI fragment from pFI793) (SEQ ID No 10).

[0190]FIG. 16 shows the nucleotide sequence of pFI913 (ie the 959 bpEcoRI/Psd fragment from pFI793) (SEQ ID No 11).

[0191]FIG. 17 is a diagrammatic representation of the outward readingprimers for pFI901 (P35 and P39), pFI911 (P32 and P36), pFI912 (P33 andP37) and pFI913 (P34 and P38).

[0192]FIG. 18 is a diagrammatic representation showing the formation ofthe 1.5 kb PCR product, using primers P34 and P39, which spans theregion in the cosmid between the inserts of pFI913 and pFI901.

[0193]FIG. 19 shows the nucleotide sequence of the merged contigspFI913/PCR product/pFI901 (4259 bp) (SEQ ID No 12).

EXAMPLE 1

[0194] Isolation and growth of Pseudomonas fluorescens biovar. V. strainAN103

[0195] Experimental

[0196] Growth media

[0197] Organisms were grown on the following media:

[0198] Minimal Medium (MM) contained, per 1: KH₂PO₄, 5 g; (NH₄)₂SO₄, 1g; FeSO₄, 0.5 mg; CaCl₂, 0.5 mg; MnCl₂.5H₂O, 5 mg; (NH₄)₆Mo₂O₇.4H₂O, 1.1mg; MgSO₄, 5 mg; EDTA, 50 mg; ZnSO₄.7H₂O, 28 mg; CuSO₄.5H₂O, 1.6 mg;CoCl₂.6H₂O, 1.6 mg. The pH was 7.0. Carbon sources were included asindicated.

[0199] Tryptone- and yeast-based medium (LBMod) contained, per l:tryptone (Bacto; Difco, Detroit, USA), 10 g; yeast extract (Bacto), 5 g;NaCl, 10 g. The pH was adjusted to 7.5. LB Medium was identical with LBMod, with the addition of glucose (1 g/l).

[0200] Solid media were prepared with the addition of agar (Difco), 15g/l.

[0201] Isolation of Pseudomonas fluorescens biovar. V, strain AN,103

[0202] The organism was isolated from surface soil, on the basis ofability to grow on trans-ferulate as sole carbon source. Initially, a Igsoil sample was added to 100 ml sterile Minimal Medium (MM), containingtrans-ferulic acid (10 mM). After 2 weeks at 25° C., with shaking at 200rpm, a sample (100 μl) was removed and added to 200 ml of fresh mediumcontaining 10 mM trans-ferulic acid; this was repeated twice. Serialdilution onto solid medium (MM) containing 10 mM trans-ferulate as solecarbon source enabled isolated colonies to be obtained which werereplica-plated onto MM plates containing individual substrates as carbonsources. Several strains able to use trans-ferulate as sole carbonsource were isolated—one (AN103), which was capable of growing also onvanillin, was selected for further work.

[0203] Growth of strain AN103

[0204] The organism was grown routinely at 25° C. on MM, with shaking,using vanillic acid (10 mM) or trans-ferulic acid (10 mM) as sole carbonsource; 50 ml of medium was used in a 250 ml Erlenmeyer flask. Growthwas monitored by measuring absorbance at 550 or 600 nm.

[0205] For long-term storage, bacteria from logarithmic-phase cultureswere centrifuged and then resuspended in Minimal Medium (MM) containing50% glycerol. They were then stored at −70° C. Cultures from thesefrozen stocks were reinitiated by transfer onto LB or LB-MOD solidmedium, followed by inoculation into liquid medium containing 10 mMtrans-ferulic acid.

[0206] Results

[0207] The organism was isolated from soil samples rich in decayedvegetation and was shown to be a strain of Pseudomonas fluorescens usingstandard identification techniques. As shown in Table I, the bacteriumwould grow not only on trans-ferulate as sole carbon source, but also onseveral closely-related compounds, including vanillate, protocatechuateand caffeate. Growth on vanillin was observed at low concentrations (<1mM) but was variable; higher concentrations were growth-inhibitory. Ifthe organism was grown on vanillate, transfer to medium containingtrans-ferulate as sole carbon source was followed by a lag in the growthcurve; this was not observed if the transfer was to medium containingboth vanillate and trans-ferulate (FIG. 2). During a growth cycle ontrans-ferulate, a transient increase in vanillate was observed at aroundthe time when trans-ferulate disappearance was maximal (FIG. 3),suggesting that vanillate was a catabolite of trans-ferulate. A smallamount of protocatechuate was also observed when the culture medium wasexamined by TLC (not shown).

[0208] Table I: Relative growth of Ps. fluorescens biovar. V, strainAN103 on a range of carbon substrates

[0209] Substrates were provided in MM Medium at 10 mM concentration(vanillin, 1 mM) and relative growth after 48 h at 25° C. was monitoredby measuring absorbance at 600 nmn. Substrates Relative Growth (%)Ferulic acid 100 Caffeic acid 79 Sinapic acid 0 Cinnamic acid 0 Vanillin<100 Vanillic acid 140 Protocatechuic acid 77 Protocatechuic aldehyde 0Glucose 221 Acetate 47 Methanol 0

EXAMPLE 2

[0210] Trans-ferulate metabolism in cell-free extracts and mechanism ofcleavage

[0211] Experimental

[0212] Chemicals

[0213] Chemicals and biochemicals were routinely obtained from SigmaChemical Co. Ltd, Poole, Dorset, UK, Aldrich Chemical Co. Gillingham,Dorset, UK or BDH-Merck, Poole, Dorset, UK. The synthesis, of CoASHthioesters is described below.

[0214] Preparation of 4-hydroxy-3-methoxyphenyl-β-hydroxypropionyl SCoA(HMPHP SCoA)

[0215] This compound was prepared starting from a Reformatskycondensation of vanillin with ethyl bromoacetate (see R. L. Shriner, TheReornatsky Reaction in “Organic Reactions”, R. Adams, W. E. Bachmann, L.F. Fieser, J. R. Johnson and H. R. Snyder, eds., vol. 1, pp 1-37, JohnWiley, New York [1942]), followed by purification of the resulting ethyl4-hydroxy-3-methoxyphenyl-β-hydroxypropionate (ethyl HMPHP) by HPLC,hydrolysis to the free acid, N-succinimidylation and, finally, exchangeof the N-succinimidyl group with CoASH and isolation of the CoASHthioester by preparative TLC (see V. Semler, G. Schmidtberg and G. G.Gross, Z. Naturforsch. 42 c, 1070-1074 [1987]).

[0216] Vanillin (3 g) was mixed with 1.9 ml of ethylbromoacetate and 2 gof dry Zn dust in 60 ml of dry 1,4-dioxane in a round-bottomed flaskfitted with drying tubes and a reflux condenser. The reaction mixturewas heated gently to boiling using a heating mantle and refluxed gentlyfor ca. 1 h. After being allowed to cool, the mixture was acidified with60 ml of 10% H₂SO₄ and extracted with 4×120 ml of diethyl ether. Thecombined ether phases were dried with anhydrous Na₂SO₄ and unreactedvanillin was removed by washing with 3×100 ml of sat. K₂S₂O₅. The etherphase was then rotary evaporated under vacuum at ca. 30° C. to removethe ether, leaving a liquid residue (ca. 10 ml). This was then appliedto a preparative C-18 reverse-phase HPLC column (Dynamax 60A, 8 μm, 250mm×41 mm; Rainin, Woburn, Mass., USA) and eluted at 12 ml min⁻¹ with agradient of MeOH/H₂O, containing 1 mM trifluoroacetic acid. [Solvent Acomprised 40% MeOH/1 mM trifluoroacetic acid; solvent B comprised 100%MeOH/1 mM trifluoroacetic acid; at time=0 min, solvent was 20% B, risinglinearly to 40% B at 28 min and 100% B at 35 min]. Fractions weremonitored by absorbance at 280 nm and material eluting between 37 and 45min was collected. The solvent was removed under vacuum at ca. 35° C.and the remaining material brought to −20° C. overnight. The precipitatewhich formed was then filtered off rapidly and freeze-dried to give 300mg of white substance. This was identified as4-hydroxy-3-methoxyphenyl-β-hydroxypropionic acid ethyl ester (ethylHMPHP) by MS [M⁻]=240 and, on alkaline hydrolysis (1M KOH; 30 min), gaverise to the free acid.

[0217] To generate the N-succinimidyl ester, 30 mg of ethyl HMPHP washydrolysed for 40 min at room temperature in 0.5 ml of 1M KOH. Oxalicacid (0.6 ml of 0.5 M) was then added to bring the pH to ca. 3-4. Thesolution was extracted successively with Et₂O (5×ca. 10 ml); the organicphases were then pooled and evaporated to dryess. N-Hydroxysuccinimide(0.1 mmol; 11.5 mg) was then added in 1.2 ml of dry 1,4-dioxane. Thiswas then followed, gradually, by 0.1 mmol (20.7 mg) ofdicyclohexylcarbodiimide in 0.6 ml of dry 1,4-dioxane. The reactionmixture was allowed to stand at room temperature for ca. 4 h and thenfiltered to remove precipitated DCU, a further 1.8 ml of dry dioxanebeing added to wash the filter.

[0218] The N-succinimidyl ester was not isolated from the reactionmixture but was converted in situ into the CoASH thioester. LithiumCoASH (40 mg; ca. 0.05 mmol) was dissolved in 2.4 ml of 0.1M NaHCO₃ andthe reaction mixture was added; the exchange reaction was performedunder N₂, with stirring, for ca. 2 h at room temperature. The pH of themixture was then adjusted to ca. 3-4 by the addition of 70 μl of 2.8 MHCl and the mixture was stored at −70° C. The CoASH thioester of HMPHPwas finally isolated by preparative TLC. Cellulose TLC plates (Avicel;1000 μm; Analtech, Newark, Del., USA), to each of which was applied 200μl of reaction mixture, were developed in nBuOH/HOAc/H₂O (5/2/3, v/v/v).The CoASH thioester was localised at R_(F) 0.4-0.5 using a short-waveu/v lamp and recovered from the plate by scraping and elution with 50%MeOH. Identification was confirmed by MS [M=960] and by hydrolysis tothe free acid (cf. ethyl HMPHP) which was measured by HPLC and usedroutinely as the basis for assay. The CoASH thioester showed anabsorption maximum at 258 nm, and lacked the absorption maximum at 345nm characteristic of trans-feruloyl SCoA. This molecule— and thecorresponding ethyl ester and free acid—carry an asymmetric centre atthe β-carbon; however, no attempt was made here to resolve the opticalisomers during or after synthesis.

[0219] Preparation of vanilloyl SCoA

[0220] Vanilloyl SCoA was produced from vanillic acid via theN-succinimidyl ester, essentially according to the method described byV. Semler, G. Schmidtberg and G. G. Gross (Z. Natursforsch. 42c,1070-1074 [1987]) for the synthesis of piperoyl SCoA.

[0221] To a stirred solution of vanillic acid (5 mmol) andN-hydroxysuccinimide (5 mmol) in 30 ml of dry 1,4-dioxane was added, insmall portions, 7.5 mmol of solid dicyclohexylcarbodiimide. The solutionwas stirred overnight at room temperature, and precipitated DCU wasremoved by filtration. The filtrate was evaporated under reducedpressure at 40° C. and the oily residue dissolved in boiling CHCl₃. TheN-succinimidyl vanillate was crystallised from solution by the dropwiseaddition of petroleum ether (b.p.: 30° C.-40° C). Approx 750 mg wasrecovered.

[0222] To generate vanilloyl SCoA from N-succinimidyl vanillate, CoASH(sodium salt; 200 mg) was dissolved in 4 ml of 0.1 M NaHCO₃.N-Succiniinidyl vanillate (120 mg in 4 ml of dioxane) was then addedgradually over a ca.40 min period at room temperature, sparging with N₂.A further 4 ml of 0.1M NaHCO₃ was then added, together with a further 8ml of dioxane. Incubation under N₂ at room temperature, with stirring,was continued for a further 1 h. The pH was then adjusted to ca. 2.8with 1M HCl and the solution was frozen and stored at −70° C. Isolationof vanilloyl SCoA was by preparative TLC, as described above with nBuOH/HOAc/H₂O(5/2/3, v/v/v) as solvent. Vanilloyl SCoA (R_(F). 0.5-0.6)was identified using a short-wave u/v lamp and recovered by scraping,elution with 40% MeOH and freezedrying. Identification was confirmed byMS ([M⁻]=916) and the thioester liberated vanillic acid on alkalinehydrolysis.

[0223] Preparation of trans-feruloyl SCoA and other cinnamoyl SCoAthioesters

[0224] Trans-feruloyl SCoA was prepared from trans-ferulic acid via theN-succinimidyl ester, as described above for vanilloyl SCoA. Finalisolation was achieved similarly by preparative TLC and elution,identification being confined by MS and by alkaline hydrolysis to freetrans-ferulic acid. Caffeoyl and p-coumaroyl-SCoA thioesters wereprepared similarly.

[0225] Preparation of cell-free extracts

[0226] Cell-free extracts of logarithmic-phase cultures (6-10 h afterinoculation) were prepared by sonication. Cells from ca. 200 ml ofmedium were pelleted by centrifugation, and resuspended in 5-10 ml ofExtraction Buffer (routinely 40 mM KPi; pH 7.2, containing 10 mMdithiothreitol). They were then sonicated (MSE Soniprep 150; FisonsInstruments, Crawley, Sussex, UK) at 4° C. (5×20 s; 22 Amplitude micronson full power), and centrifuged (20 000 x g; 20 min; 4° C.). Extractswere routinely stored frozen at −70° C. and in some instancesbuffer-changed using a PD10 column (Pharmacia) before use. The proteincontents of extracts were variable—between 0.25 and 1.8 mg/ml.

[0227] Incubation of cell-free extracts

[0228] Cell-free extracts were routinely incubated at 30° C. and pH 7.5in a reaction mixture (1 ml) containing 90 mM Tris HCl buffer and 2.5 mMMgCl₂, together with (as appropriate) 0.5 mM trans-ferulic acid, 0.2 mMCoASH (Li salt) and 2.5 mM ATP. This complete reaction mixtureconstituted an assay for trans-ferulate: CoASH ligase, where the initialincrease in absorbance at 345 nm was monitored against a blank reactionmixture from which CoASH was omitted. Incubations with HMPHP SCoA(generally 0.4 mM) were performed similarly, but with the omission oftrans-ferulic acid, CoASH and ATP.

[0229] Vanillin: NAD⁺ oxidoreductase was assayed at 30° C. and pH 7.0 bymonitoring the initial decrease in absorbance at 340 nm against a blankcuvette from which NAD⁺ was omitted. Because of the similarity inextinction coefficient at 340 nm for vanillin and for NADH, thesensitivity of the assay was increased by catalysing the regeneration ofNADH to NAD⁺ by providing lactate dehydrogenase and pyruvate. Reactionmixtures contained, in 1 ml volume, 75 mM KPi buffer, pH 7.0, 0.125 mMvanillin, 1.2 mM Na pyruvate, lactate dehydrogenase (rabbit muscle), 1.1U and NAD⁺, 0.5 mM.

[0230] HPLC analysis

[0231] Metabolites of trans-ferulic acid, including the CoASHthioesters, were analysed and quantitated by HPLC using a LichrosorbRP-18 column (20 cm×4.6 mm; Capital HPLC, Broxburn, West Lothian, UK)with a multiphasic gradient; solvent “A” was 20 mM NaOAc, adjusted to pH6.0 and solvent“B” was MeOH; the flow rate was 1.2 ml/min; theproportion of solvent “B” rose linearly from 0% at 0 min to 10% at 15min and thence to 50% at 40 min and 70% at 45 min, finally decreasing to0% at 50 min. Detection was with a Spectra Focus detector (ThermoSeparation Products, Stone, Staffs. UK), which permitted u/v spectralanalysis of each eluting component.

[0232] Typical approximate retention times were: CoASH, 3 min; vanillicacid, 7 min; trans-ferulic acid, 19 min; acetyl SCoA, 22 min; HMPHPSCoA, 29 min; vanilloyl SCoA, 31 min; vanillin, 31.5 min; trans-feruloylSCoA, 34 min.

[0233] Mass spectrometry

[0234] Mass spectra (+−ve and −−ve ion) were recorded on a MS 9/50 massspectrometer (Kratos Instruments, Manchester UK), using xenon fast atombombardment (FAB) at a potential of 5-7 kV using glycerol as matrix (seeG. R. Fenwick, J. Eagles and R. Self, Biomedical Mass Spectrometry 10,382-386 (1983)).

[0235] Protein assay

[0236] Protein was assayed by the method of M. M. Bradford (Anal.Biochem. 72, 248-254 (1976)), using Bio-Rad dye reagent (Bio-RadLaboratories, Richmond, Calif., USA) and bovine serum albumin asstandard.

[0237] Results

[0238] Crude extracts of Ps. fluorescens biovar. V, strain AN103, fromcells grown on vanillate together with trans-ferulate, were able toproduce vanillate when supplied with trans-ferulate, CoASH, ATP, Mg²⁺ions and NAD⁺. In the absence of NAD⁺, vanillate was not formed andvanillin accumulated in its place (FIG. 4). The quantity of vanillinformed in the absence of NAD⁺ was smaller than the amount of vanillateformed in its presence. Essentially no vanillin accumulated in thepresence of NAD⁺.

[0239] This utilisation of trans-ferulate by crude extracts wasdependent upon CoASH and ATP and partially upon Mg²⁺ ions (Table II).These properties indicate an initial activation of trans-ferulate totrans-feruloyl SCoA by trans-ferulate: CoASH ligase. This was furthershown by the rapid development of a CoASH-dependent absorbance maximumat 345 nm and particularly by a transient bathochromic shift, causingthe appearance of a yellow colour, if the reaction mixture was madealkaline with NaOH (data not shown). In the initial stages of theoverall reaction, the linear increase in absorbance at 345 nm enabledthe activity of trans-ferulate: CoASH ligase to be assayed directly.During the later stages, however, absorbance at 345 nm would becontributed by both trans-feruloyl SCoA and vanillin or, in the presenceof NAD⁺, NADH, each of which has substantial absorbance at thiswavelength.

[0240] Table II: Cofactor requirements for trans-ferulate utilisation byP. fluorescens biovar. V, strain AN103 cell-free extracts

[0241] Reaction mixtures (165 μg protein) were incubated for 4 h at 30°C. as described in Experimental, with omissions from the completereaction mixture as indicated. Reaction products (nmol) ReactionTrans-ferulate Mixture (remaining) Vanillin Vanillate Complete 267 n.d.311 —CoASH 550 n.d. 40 -NAD⁺ 258 228 23 -ATP 513 n.d. 33 —Mg²⁺ 425 n.d.153

[0242] An overall non-oxidative cleavage of trans-feruloyl SCoA isimplied in FIG. 5 which shows, in the absence of NAD⁺, an equivalencebetween the formation of vanillin and that of acetyl SCoA. (Theformation of [2-¹³C] acetyl SCoA from trans-ferulate ¹³C-labelled in theβ-carbon atom was also confirmed by NMR spectroscopy (not shown).) Thecleavage mechanism was investigated further by synthesising chemicallythe hydrated derivative of trans-feruloyl SCoA,4-hydroxy-3-methoxy-phenyl-β-hydroxypropionyl SCoA (HMPHP ScoA). Thiswas incubated with cell-free extract and shown to be converted rapidlyto acetyl SCoA and vanillin, in equimolar proportions (FIG. 6). A smellof vanillin was obtained when HMPHP SCoA was used as a substrate. Thishydrated intermediate was not only metabolised in the forward direction,however, since an almost equivalent back reaction to fernloyl SCoA(putatively trans) was also observed. The rapidity of utilisation ofHMPHP SCoA was consistent with the failure to observed its accumulation,using HPLC, during cell-free incubations with trans-ferulate, CoASH, ATPand Mg²⁺ ions. This cleavage of HMPHP SCoA in the absence of NAD⁺indicated no intervening β-oxidation to the β-keto thioester(4-hydroxy-3-methoxybenzoyl) acetyl SCoA (cf. M. H. Zenk, Anal. ZPflanzenphysiol 53, 404-414 (1965)). Attempts to prepare this compoundfor cell-free studies were unsuccessful, but its expected cleavageproduct, vanilloyl SCoA, was prepared and shown not to be metabolised tovanillin by cell-free extracts in the presence of NADH, even whensimultaneous incubations with trans-ferulate in the absence of NAD⁺actively produced vanillin.

[0243] Besides trans-ferulic acid, caffeic acid and p-coumaric acidswere converted to thioesters of CoASH by crude extracts of Ps.fluorescens biovar. V, strain AN103 (Table III).

[0244] Table III. Formation of CoASH thioesters oftrans-p-hydroxycinnamic acids by crude extracts of Ps. fluorescensAN103. The activity was assayed spectrophotometrically as described fortrans-ferulate: CoASH ligase, measuring the initial rate of increase inabsorbance at 345 nm in the case of trans-feruloyl SCoA formation, andat the corresponding absorbance maxima for the other SCoA thioesters.Substrate Activity (nkat/mg protein) Ferulate 0.50 Caffeate 0.39p-Coumarate 0.37

EXAMPLE 3

[0245] Mutants in trans-ferulate metabolism

[0246] Experimental

[0247] Mutagenesis of Pseudomonas fluorescens biovar. V, strain AN103

[0248] Ethyl methanesulphonate (EMS) was used for mutagenesis. Bacteriawere grown for 2 d at 25° C. in minimal medium (MM) with vanillic acidas carbon source; 1 ml of culture was then inoculated into 50 ml ofLB-MOD and grown for 16 h at 25° C. The cells were centrifuged andresuspended in 0.M KH₂PO₄ (1.25 ml) to give a cell density of 4×10⁹cells/ml (OD₅₈₀ of 1.0=6×10⁷ cells/ml). An aliquot of this cellsuspension was serially diluted (10⁻²-10⁻⁸) and plated onto LB-MODplates (0.1 ml per plate) to provide control cell counts for assessmentof the efficiency of mutagenesis.

[0249] A cell suspension (1 ml) was incubated with 0.08 ml of EMS in atotal of 3 ml of 0.1M KH₂PO₄ at 37° C. for 45 min. The cells were thenprecipitated by centrifugation at 4° C. and the cell pellet was washedtwice with 10 ml of LB-MOD medium, prior to resuspension in 1 ml of thismedium. An aliquot was serially diluted (10⁻²-10⁻⁸) and plated ontoLB-MOD plates; these were incubated at 25° C. overnight, together withthe plates of the unmutagenised cells, to obtain an estimate of kill(70% kill indicates efficient mutagenesis). The remaining mutagenisedcells (0.9 ml) were inoculated into LB-MOD medium (50 ml) and incubatedovernight at 25° C. The mutagenised cells were then enriched for mutantsin trans-ferulate utilisation by treatment with carbenicillin in minimalmedium (MM) in the presence of trans-ferulic acid. The cells wereharvested by centrifugation at 4° C., washed twice with MM (10 ml) andresuspended in 20 ml of MM. A sample (1 ml) was inoculated into MM (15ml) and incubated at 25° C. for 1 h; then trans-ferulic acid (10 mMfinal concentration) and carbenicillin (2 mg/ml final concentration)were added. A control flask was prepared containing trans-ferulic acid,but not carbenicillin. Both flasks were incubated overnight at 25° C.for 16 h, monitoring OD₅₈₀ to estimate growth and confirm theeffectiveness of the antibiotic. Penicillinase (10 units) was then addedto destroy the carbenicillin, incubating overnight at 25° C. The cellswere harvested by centrifugation at 4° C., washed twice in MM (10 ml)and resuspended in 5 ml of MM; 1 ml of these resuspended cells were theninoculated into 50 ml of MM containing 10 mM vanillic acid and incubatedat 25° C. for ca. 24 h.

[0250] These enriched cells were screened by replica-plating for mutantsunable to use trans-ferulic acid. The enriched stock was diluted to10⁻⁶, plated onto LB-MOD (0.1 ml per plate), incubated at 25° C. for 2 dand then replica-plated onto MM containing 10 mM vanillic acid or 10 mMtrans-ferulic acid. The plates were incubated at 25° C. for 2-3 d andscreened for colonies able to grow on vanillate but unable to grow ontrans-ferulate.

[0251] Results

[0252] By mutagenesis of strain AN103 with ethyl methane sulphonate, twoclasses of mutants unable to utilise trans-ferulate as sole carbonsource were isolated; these were van 1, van 2 and van 3 and, secondly,van 10 and van 11. Following growth on vanillate plus trans-feralate, arepresentative of the first of these, van 1, showed no activity incell-free incubations with either trans-ferulate or vanillin and lackedboth trans-ferulate: CoASH ligase and the enzyme that converts vanillinto vanillate, vanillin: NAD⁺ oxidoreductase. In contrast, the typerepresentative of the second class, designated van 10, possessed levelsof activity of both trans-ferulate: CoASH ligase and vanillin: NAD⁺oxidoreductase similar to those found in strain AN103, but in thepresence of NAD⁺ generated very little vanillate (Table IV). Cell-freeextracts of van 10 were examined further for their ability to metaboliseHMPHP SCoA. They metabolised this thioester actively, but appearedpredominantly to dehydrate it to feruloyl SCoA; vanillin formation wassubstantially inhibited in comparison to the AN103 extract (Table V).These observations suggested that van 1 was a regulatory mutant,defective in its induction by trans-ferulate, whilst van 10 appeared tobe defective in the HMPHP ScoA cleavage activity.

[0253] Table IV: Trans-ferulate metabolism in cell-free extracts of P.fluorescens biovar. V, strain AN103 and of mutant strains van 1 and van10.

[0254] Cells were grown for 6 h in MM medium containing 10 mM vanillatetogether with 10 mM trans-ferulate. Extracts were then prepared as inExample 2 and assayed for trans-ferulate:CoASH ligase and vanillin:NAD⁺oxidoreductase. Extracts (ca. 0.3 mg of protein) were also incubated for4 h in the presence of NAD⁺ to determine relative amounts of vanillateformed. Enzyme activity (nkat mg⁻¹ protein) Ferulate: CoASH Vanillin:NAD Vanillate formed Strain ligase oxidoreductase (nmol mg⁻¹ protein)AN103 1.7 1.2 807 van 1 0 ˜0.05 0 van 10 2.0 1.4 59

[0255] Table V: Utilisation of HMPHP SCoA by extracts of Ps. fluorescensbiovar. V, strains AN103 and van 10

[0256] Extracts (AN103, 14 tg protein; van 10, 68 μg protein) wereincubated at 30° C. for 7 min in 1 ml vol containing 0.3 mM HMPHP SCoA.The increase in absorbance at 345 nm was measured against a blankreaction mixture containing no extract. Vanillin formation was measuredby HPLC; the production of feruloyl SCoA was in this instance calculatedfrom the increase in absorbance at 345 nm, after subtraction of thecontribution from vanillin. Feruloyl SCoA Vanillin Strain ΔA₃₄₅ (nmol)(nmol) AN103 0.75 18.4 23.8 van 10 0.63 29.2 4.5

EXAMPLE 4

[0257] Induction of trans-ferulate metabolism in strain AN103 andpurification of HMPHP SCoA cleavage enzvme

[0258] Experimental

[0259] Purification of trans-feruloyl SCoA hydrataselaldol cleavageenzyme

[0260] Cells (from 21 of culture grown for 72 h on MM with 10 mMtrans-ferulic acid as substrate; OD₅₆₅ca.=0.5) were extractedessentially as described in Example 2 to give 50 ml of crude extract,containing 1.28 mg of protein/ml.

[0261] Extract (16 ml, diluted to 40 ml), was applied at roomtemperature and 2 ml/min to a Mono Q HR10/10 anion-exchange column(Pharmacia, Piscataway, N.J., USA), preequilibrated with 20 mM Trisbuffer (pH 7.5) containing 10 mM dithiothreitol. After elution ofunadsorbed protein, protein bound to the column was eluted with a lineargradient of increasing NaCl concentration: from 0 to 0.5M NaCl in 100 mlof buffer.

[0262] Fractions eluting between 0.18 and 0.3 M NaCl and containingactivity with HMPHP SCoA, as determined using the microtitre plate assay(see below), were pooled and buffer-changed by dialysis into 25 mMbis-Tris buffer, containing 10 mM dithiothreitol and adjusted to pH 7.1with iminodiacetic acid. They were then applied at 0.75 ml/min to a MonoP HR 5/20 chromatofocusing column (Pharmacia), preequilibrated with thesame buffer. After eluting unadsorbed protein from the column, adsorbedprotein was eluted with a gradient of decreasing pH, generated byapplying 46 ml of 10% (v/v) Polybuffer 74 (Pharmacia), containing 10 mMdithiothreitol and adjusted to pH 4.0 with iminodiacetic acid. Activitywith HMPHP SCoA was eluted between pH 5.5 and 5.1.

[0263] The active fractions were again pooled together, andbuffer-changed into 20 mM Tris buffer (pH 7.5), containing 1.7 M(NH₄)₂SO₄ and 10 mM dithiothreitol, using PD10 columns (Pharmacia),before application at 0.5 ml/min to a PhenylSuperose HR 5/5 hydrophobicinteraction chromatography column (Pharmacia) preequilibrated with thisbuffer. Elution of bound protein was achieved with a decreasing gradientof (NH₄)₂SO₄ in buffer, from 1.7 M to zero over 30 ml and thencontinuing with buffer alone for a further 5 ml. Activity with HMPHPSCoA was eluted in this final 5 ml of buffer.

[0264] At each stage of purification, active fractions were detected bya micro-adaptation of the assay with HMPHP SCoA described above (Example2); reactions were performed in 100 μl of reaction mixture for ca. 4 minat room temperature in microtitre wells and absorbance was then measuredin an MR 5000 microtitre plate reader (Dynatech, Guernsey, ChannelIslands), equipped with a 340 nm filter. The activity of the pooledfractions was measured using HPLC to determine the reaction products ofboth HMPHP SCoA, (0.4 mM) and trans-feruloyl SCoA (0.28 mM) assubstrates. Reaction mixtures containing 10 μl of enzyme were incubatedin 100 μl volume as described above; the reaction was terminated with100 μl of acidified MeOH (pH 3) after 2 min (HMPHP SCoA) or 5 min(trans-feruloyl SCoA) of incubation at 24° C. The proportionality of thereactions with time and with quantity of enzyme was established inpreliminary determinations.

[0265] Samples (10 μl) of enzyme at each stage of purification wereanalysed by SDS-PAGE, with Coomassie or silver staining, essentially asdescribed by H. Schägger and G. von Jagow, Anal. Biochem. 166, 368-379(1987). An Atto AE6450 gel apparatus was used (supplied by GeneticResearch Instrumentation, Dunmow, Essex, UK).

[0266] Electroelution of protein bands from fixed, stained gels wasperformed using a Bio-Rad Model 422 electroeluter according to themanufacturer's directions. Eluted protein was then deposited bycentrifugation onto a Pro-Spin membrane (Applied Biosystems, FosterCity, Calif., USA) used in accordance with the manufacturer'srecommendations). N-Terminal sequencmg was performed by Alta Bioscience,University of Birmingham, Birmingham, UK.

[0267] Results

[0268] The time-course of induction of trans-ferulate:CoASH ligase instrain AN103 is shown in FIG. 7. Following transfer of vanillate-growncells to medium containing trans-ferulate, the specific activity of theligase in extracts increased approximately linearly over an 8-hourperiod. An essentially identical time-course was obtained if the cellswere transferred instead to medium containing both trans-ferulate andvanillate. Vanillate, a catabolite of trans-ferulate, therefore did notinhibit the induction process.

[0269] Induction of the capacity to grow on a different substraterepresents a significant shift in primary metabolism, which in principlemight be detectable by electrophoresis of a crude protein extract.Cell-free extracts analysed by SDS-PAGE, with Coomassie staining (seeExperimental), did indeed show a distinct difference in protein bandingbetween trans-ferulate-grown cells and vanillate-grown cells (FIG. 8).Extracts from trans-ferulate-grown cells exhibited a new, or verystrongly enhanced, band corresponding to a polypeptide of molecularweight ca. 31 kD.

[0270] N-Terminal amino acid sequencing of this polypeptide, followingits removal from the gel by electroblotting, gave the followingsequence:Ser-Thr-Tyr-Glu-Gly-Arg-Trp-Lys-Thr-Val-Lys-Val-Glu-Ile-Gln-Asp-Gly-Ile-Ala-Phe(SEQ ID No 13).

[0271] Purification of HMPHP SCoA-utilising activity was achieved byFast Protein Liquid Chromatography (FPLC—Pharmacia). As described above,fractions were screened for activity with HMPHP SCoA using a microtitreplate scanner; active fractions were then pooled together and activitywas then measured both with HMPHP SCoA and with trans-feruloyl SCoA,determining the reaction products by HPLC.

[0272] The results of the purification are given in Tables VI and VII.Vanillin and acetyl SCoA were produced in approximately equimolaramounts, throughout the purification, with either HMPHP SCoA ortrans-feruloyl SCoA as substrate. There was an approximatecopurification of the activities with trans-feruloyl ScoA and HMPHP SCoAas substrate, including the formation of feruloyl SCoA from HMPHP SCoA(dehydratase reaction; reverse of reaction II): vanillin-formingactivity from trans-feruloyl SCoA (reactions II+III) was purified11.5-fold, vanillin-forning activity from HMPHP SCoA (reaction III) waspurified 11.7-fold and the dehydratase reaction (reverse of reaction H)was purified 9.1-fold. Approximately 20-25% of each of these activitieswas fmally recovered.

[0273] Table VI: Purification of trans-feruloyl SCoA hydratase/aldolcleavage enzyme.

[0274] Purification from cells of Ps. fluorescens biovar V, strainAN103, was undertaken as described in Experimental, measuringactivity—vanillin as product—with both trans-feruloyl SCoA and HMPHPSCoA as substrates. Values in parentheses show activity measuring acetylSCoA as product. Part 1 Total activity Specific activity (nkat)(nkat/mg) trans- Total trans- Purification feruloyl HMPHP proteinferuloyl HMPHP stage SCoA SCoA (mg) SCoA SCoA Crude 85.2 59.2 20.5 4.162.89 Extract (68.2) (54.9) Mono Q 80.6 45.1 4.37 18.4 10.3 Fractions(66.2) (42.9) Mono P 56.9 35.4 1.61 34.1 21.2 Fractions (51.9) (33.4)Phenyl 22.1 15.5 0.46 48.0 33.7 Superose (19.8) (15.1) Fractions

[0275] Part 2 Purification Recovery (fold) (%) trans- trans-Purification Ratio of feruloyl HMPHP feruloyl HMPHP stage activitiesSCoA SCoA SCoA SCoA Crude 1.44 1.00 1.00 100 100 Extract Mono Q 1.794.42 3.56 94.6 76.2 Fractions Mono P 1.61 8.20 7.34 66.8 59.8 FractionsPhenyl 1.42 11.5 11.7 25.9 26.2 Superose Fractions

[0276] Table VII: HMPHP SCoA dehydratase activity during purification oftrans-feruloyl SCoA hydratase/aldol cleavage enzyme

[0277] Conditions and other data as Table VI. Dehydratase reactionmeasured as feruloyl SCoA production. Ratio to Total Specific HMPHP SCoAPurification activity activity Purification Cleavage stage (nkat)(nkat/mg) (fold) Activity* Crude 171 8.34 1.00 2.89 Extract Mono Q 12127.7 3.32 2.69 Fractions Mono P 101 60.5 7.25 2.85 Fractions Phenyl 34.975.9 9.10 2.25 Superose Fractions

[0278] SDS-PAGE of the combined active fractions at each stage revealedthe enhancement of a 31 kD protein band (FIG. 8), indicatingpurification to apparent homogeneity after chromatography onPhenylSuperose (Pharmacia). This band co-migrated with the bandassociated with growth of strain AN103 on trans-ferulate and gave thesame N-terminal amino-acid sequence: Ser-Thr-Tyr-Glu-Gly-Arg-Trp (SEQ IDNo 14).

[0279] Definitive proof of the catalysis of both reactions II and III bythis protein was achieved as a result of expression of the gene inEscherichia coli. (see Example 5).

[0280] The Mono-P-purified enzyme was able to accept, as alternativesubstrates to trans-feruloyl SCoA, trans-caffeoyl SCoA andtrans-4-coumaroyl SCoA (Table VIII).

[0281] Table VIII. Utilisation of trans-p-hydroxycinnamoyl SCoAthioesters by trans-feruloyl SCoA hydratase/aldol cleavage enzyme.Activity was determined at 30° C. and with 0.4 mM substrate using enzymefrom Ps. fluorescens AN103 (2.8 μg of enzyme protein, partially purifiedby Mono-Q and Mono-P chromatography) as described in Experimental.Substrate Activity (nkat/mg of protein) Feruloyl SCoA 0.60 Caffeoyl SCoA0.36 p-Coumaroyl SCoA 0.72

EXAMPLE 5

[0282] Isolation of the genes required for the conversion oftrans-feruloyl SCoA to vanillic acid (vanillate) in Pseudomonasfluorescens strain AN103

[0283] A strain of Pseudomonas fluorescens (biovar. V, AN103) wasisolated from soil at the Institute of Food Research, NorwichLaboratory, which was able to grow on trans-ferulic acid converting itto vanillic acid via vanillin. The proposed biochemical pathway for theconversion of trans-ferulic acid to vanillic acid shown in FIG. 1 wassubstantiated in the experiments described above in Experiments 2-4.

[0284] In order to clone the genes required for the conversion oftrans-feruloyl SCoA to vanillic acid the strategy of complementingmutant derivatives of Ps. fluorescens AN103 that were unable to grow ontrans-ferulate as sole carbon source was used. The isolation andcharacterization of mutants is described above in Example 3 and mutantsvan10 and van11 were used for clone isolation. As described in Example3, these mutants appeared to be defective in a gene involved in theconversion of trans-feruloyl SCoA to vanillin.

[0285] A genomic library of Ps. fluorescens AN103 DNA was prepared inthe mobilisable cosmid cloning vector pLAFR3 (B. Staskawicz, D.Dahlbeck, N. Keen, and C. Napoli, J. Bact. 169, 5789-5794 (1987)).Genomic DNA was isolated from Ps. fluorescens AN103 and partiallydigested with Sau 3A1 at 37° C. for 7-10 min. The DNA was thensize-fractionated on a NaCl gradient (1.25-5M). The fraction containingDNA of 2040 kb was selected and 0.5 μg ligated into the dephosphorylatedBam H1 site of the broad- host-range cosmid cloning vector, pLAFR3. Onehalf of the ligation mix was packaged into bacteriophage lambdaparticles using a Gigapack II XL kit (Stratagene, La Jolla, Calif.,USA). The packaged cosmids were transfected into Escherichia coli strain803 (W. B. Wood, J. Mol. Biol. 16, 118-133 (1966)). Approximately 10,000primary transfectants were obtained. The lawn of cells obtained waswashed from the selection plates and glycerol-containing stocks preparedfor storage at −70° C.

[0286] The genomic library of Ps. fluorescens AN 103 DNA in cosmidpLAFR3 was introduced into the two mutant Ps. fluorescens derivativestrains van10 and van11 using the helper plasmid, pRK2013 (D. Figurskiand D. R. Helinski, Proc Natl. Acad. Sci. USA 76, 1648-1652 (1979)). Themutant strains were inoculated into minimal medium MM containing 10 mMvanillic acid and incubated at 25° C. for 2 days. The Escherichia colistrain carrying the helper plasmid (E. coli 803pRK2013) was inoculatedinto LB-Mod medium (10 ml) and incubated at 37° C. for 6 h. At the sametime, 0.1 ml of the glycerol-containing stock of the AN103 genomiclibrary was similarly inoculated and incubated. The growth of all threecultures was monitored by measuring OD₆₀₀ and appropriate volumescombined in a centrifuge tube to give equal populations of the threeorganisms. The mixture of cells was centrifuged, resuspended in aminimal volume of the supernatant solution and spread over a sterilegridded cellulose nitrate membrane filter (47 mm diam., Whatman,Maidstone, Kent, UK) on a moist LB-Mod agar plate. The suspension wasallowed to air-dry onto the filter for a few minutes and then incubatedovernight at 25° C. The bacteria were then washed from the filter using2 ml of H₂O and aliquots (0.1 ml) were applied to selection platesconsisting of MM agar with 10 mM vanillic acid and 5 μg/ml tetracycline.These were incubated at 25° C. for 2 days and the colonies obtained(>1000 per plate) were replica-plated to similar plates containingtrans-ferulic acid in place of vanillic acid; these were incubatedsimilarly. Colonies (2-3 per plate) able to grow on the platescontaining trans-ferulic acid were selected and inoculated into fresh MMmedium containing 10 mM trans-ferulic acid and 5 μg/ml tetracycline.Four such isolates in which the mutation in the Ps. fluorescens strainsvan10 and van11 was complemented by the introduced cosmid were selectedfor further analysis. These strains were purified and the cosmid DNA wasextracted by the mini-preparation method of F. G. Grosveld, H. H. M.Dahl, E. Deboer and R. A. Flavell (Gene 13, 227-231 (1981)). The cosmidDNA was transformed into E. coli strain 803 and was again isolated asdescribed by D. S. Holmes and M. Quigley (Anal. Biochem. 114, 193-197(1981)). Two of the cosmid clones, pFI 793 and pFI 794, were isolated ascomplementing Ps. fluorescens mutant strain van 10, whereas cosmidclones pFI 795 and pFI 796 complemented Ps. fluorescens mutant strainvan 11.

[0287] To test whether the plasmid clones pFI 793, pFI 794, pFI 795 andpFI 796 would complement any of the other Ps. fluorescens mutants, eachplasmid was introduced into Ps. fluorescens mutant strains van1, van2,van3, van10 and van11 . As described above in Example 3 the mutantstrains van1, van2 and van3 appear to be defective in a regulatoryfunction that eliminates at least two different enzyme activities. ThePs. fluorescens van mutants were grown on MMO+10 mM vanillic acid agarmedium for two days at 25° C. The strain carrying the helper plasmid(803pRK2013) was grown on LB-Mod agar+kanamycin (25 μg/ml) at 37° C.overnight. The E. coli 803 cosmid clones carrying pFI 793, pFI 794, pFI795 and pFI 796 were grown on LB-Mod agar medium+ tetracycline (5 μg/ml)at 37° C. overnight. The bacteria were,patch mated (ie a loopful ofdonor, recipient and helper strain were mixed together on LB-Mod agarand incubated overnight at 25° C). The bacteria were replica plated ontoselection medium (MMO+10 mM trans-ferulic acid+ tetracycline and MMO+10mM vanillic acid+tetracycline) and incubated at 25° C. for two days. Allfour cosmid clones pFI 793, pFI 794, pFI 795 and pFI 796 complementedall of the van mutants (van1, van2, van3, van10 and van11) enabling themto grow on trans-ferulate as sole carbon source.

[0288] The cosmid clone DNAs were analyzed by digestion with restrictionendonucleases Hind III and Eco RI to reveal insert DNA. Each of the fourclones pFI 793, pFI 794, pFI 795 and pFI 796 carried inserts of between20 and 30 kb. The four cosmid clones gave distinct restriction patterns,but appeared to share some restriction fragments of the same size. Toidentify restriction fragments that were common to the cosmid clonescosmid pFI 793 was used as a probe against DNA of all four cosmid DNApreparations that had been double-digested with restrictionendonucleases EcoRI and PstI. Cosmid DNA was isolated using Qiagen midicolumns according to the manufacturers instructions and was digestedwith restriction endonucleases EcoRi and PstI. The resulting fragmentswere separated by agarose gel electrophoresis and Southern blotted to aHybond-N filter as described by Sambrook et al (Sambrook, J., Fitsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd edition,Cold Spring Harbor, N.Y., 1989). Cosmid pFI 793 DNA was linearised,denatured and labelled with digoxygenin prior to probing the Southernblotted DNA according to the instructions supplied by Boehringer (Lewes,Sussex, UK). The pFI 793 probe hybridised to all of the EcoRI/PstIrestriction fragments of pFI 795 indicating that these two clones areidentical. Excluding the vector band at least six EcoRI/PstI fragmentsappeared to be common to pFI 793, pFI 794, pFI 795 and pFI 796. Thesewere fragments of 6.6 kb, 2.9 kb, 1.8 kb, 1.4 kb, 1.25 kb and 1.1 kb.The 1.25 kb fragment appeared to be a doublet or triplet. The DNAfragment patterns of the four cosmid clones after restriction digestionwith EcoRI/PstI are shown in FIG. 9. For clarity the ethidium bromidestained DNA bands have been diagrammatically superimposed on theoriginal agarose gel.

[0289] As described above in Example 4 protein analysis of Ps.fluorescens biovar. V, strain AN103 showed that cells grown ontrans-ferulate contained much larger amounts of a protein of about 31 kDcompared with cells grown on vanillic acid. The twenty N-terminal aminoacids of this protein were sequenced. This amino acid sequence was thenused to design degenerate oligonucleotide primers that enabled the 60 bpsequence of DNA coding for this N-terminus to be amplified from pFI 793by PCR (FIG. 10). This sequence was used to probe EcoRI/PstI digests ofthe cosmid clones. By this technique the fragment containing the regionof DNA encoding the 31 kD protein in each of the cosmid clones could beidentified (FIG. 11). This proved to be a fragment of 6 kb in cosmidspFI 793 and pFI 795, a fragment of 4.3 kb in cosmid pFI 794 and afragment of 5.5 kb in cosmid pFI 796.

[0290] The 4.3 Kb EcoRI/PstI fragment of pFI 794 was sub-cloned into theE. coli vector pUC19 using strain XLI (Blue) and its nucleotide sequencewas determined using an Applied Biosystems DNA Sequencer (Model 373;Perkin Elmer, Warrington, UK); together with the manufacturer's TaqDyeDeoxy Terminator Cycle sequencing kit. A primer walking strategy wasused with oligonucleotide primers being synthesized on an ABI 392Synthesizer (Perkin Elmer, Warrington, UK). The DNA sequence of the 4.3kb fragment is presented in FIG. 12. The open reading frame encoding thesame amino-terminus as determined previously starts at position 2872 andis terminated by a stop codon at position 3700. This ORF of 828 bpencodes a protein 276 amino acids long with a molecular size of 31.010kD in good agreement with the protein gel analysis. The translatedamino-acid sequence of this gene is also presented in FIG. 12. In orderto confirm the function of this gene it was sub-cloned and expressed inE. coli. From the DNA sequence PCR primers were designed to amplify thegene such that it was flanked by restriction endonuclease sites EcoRIand BamHI. The amplified gene retained its native ribosome binding sitebeing initiated at base −29 and ending 6 bp downstream of the stopcodon. The amplified fragment was cloned into the equivalent sites ofthe E. coli expression vector pSP72 (Promega, Southampton, UK) andtransformed into E. coli JM109(DE3).

[0291] The E. coli 803 clones carrying the hydratase/cleavage enzymegene, plus a putative promoter, as a PCR product in the vector pRK415were used in a triparental patch mating experiment essentially asdescribed earlier in relation to complementation by the cosmid clonespFI 793-6. The complemented van 10 strain was demonstrated to haverecovered the ability to grow on trans-ferulic acid, confirming directlythat the mutation in van 10 resided in the gene encoding thetrans-feruloyl ScoA hydratase/cleavage enzyme.

[0292] The presence of a novel enzyme activity (cf. Example 4) in the E.coli clone was demonstrated. E. coli cells were grown at 37° C. for 3 hin 50 ml of L medium, containing ampicillin (50 μg/ml) with and withoutinduction by IPTG. Extracts were prepared as described above in Example2 for Ps. fluorescens AN103, but without centrifugation. The crudeextract was used for assay. Enzyme activity with both HMPHP SCoA andtrans-feruloyl ScoA was determined as described above in Example 4 usingHPLC to determine the reaction products. The results presented in TableIX clearly demonstrate that vanillin and acetyl SCoA were produced inequimolar proportions both with trans-feruloyl SCoA and with HMPHP SCoAas substrates. In addition, HMPHP SCoA was also dehydrated to feruloylSCoA, putatively trans-feruloyl SCoA, demonstrating the reverse ofactivity II. These results are closely similar to those obtained withthe vanillin-forming cleavage enzyme purified from Pseudomonasfluorescens AN103, although the ratio of activities with trans-feruloylSCoA and HMPHP SCoA differs slightly between the two preparations. Therewas no activity with either trans-feruloyl SCoA or HMPHP SCoA inextracts of an unmanipulated E. coli strain, whether induced or not. Inthe manipulated strain E. coli 1039 that expresses the Pseudomonas genethe specific activity was slightly lower in the exLract made followinginduction than in that made from uninduced bacteria. Since the assaymeasures only active enzyme it is conceivable that increased proteinexpression occurs upon induction but this may result in incorrectlyfolded and therefore inactive enzyme. It was not possible to detectexpression of the 31 kD protein visually on Coomassie-stained,one-dimensional SDS gels because of its co-migration with the stronglyexpressed β-lactamase encoded by the vector ampicillin resistancemarker.

[0293] Table IX: Expression of trans-feruloyl SCoA hydratase/aldolcleavage enzyme in Escherichia coli.

[0294] Enzyme was extracted as described in Example 2 and activitydetermined as described in example 4, using trans-feruloyl SCoA (0.28mM) and HMPHP SCoA (0.4 mM)) as substrates. Reaction mixtures containedca. 10 μg of protein.

[0295] n.d.—not detectable TABLE IX Expression of trans-feruloyl SCoAhydratase/aldol cleavage enzyme in Escherichia coli. Enzyme wasextracted as described in Example 2 and activity determined as describedin Example 4, using trans-feruloyl SCoA (0.28 mM) and HMPHP SCoA (0.4mM)) as substrates. Reaction mixtures contained ca. 10 μg of protein.n.d. - not detectable Specific activity (nkat/mg of protein)trans-Feruloyl SCoA as substrate HMPHP SCoA as substrate E. coliVanillin Acetyl SCoA Vanillin Acetyl SCoA Feruloyl SCoA Cell lineformation formation formation formation formation Control n.d. n.d n.d.n.d. n.d. Control n.d. n.d. n.d. n.d. n.d. (induced) 1039 1.53 1.80 1.521.80 3.35 1039 (induced) 1.24 1.34 1.27 1.46 3.16

[0296] The DNA downstream of the gene encoding the 31 kD protein wastargeted for cloning and sequencing and for analysis of additional openreading frames. A PCR-generated probe was used to identify anoverlapping XhoI fragment of 1.5 kb. Sequencing from this fragment andsubsequently directly from the parent cosmid clone pFI794 revealed asecond open reading frame of 1449 bp beginning at base 3804. Thetranslation of this nucleotide sequence revealed a polypeptide of 483amino acids. Comparison with sequences in the databases revealedappreciable homology to salicylaldehyde: NAD⁺ oxidoreductase.

[0297] In order to confirm the function of this gene, expression wasdetermined in E. coli strain DH5, which contained the vector pUC18 intowhich the full-length open reading frame had been inserted such thatexpression was from the lac promoter on the vector. Vanillin: NAD⁺oxidoreductase activity was confirmed and was absent from a controlstrain bearing the unmodified pUC 18 vector. Using the enzyme assaydescribed in Example 2, activity with vanillin as substrate wasdetermined as 3.0 nkat/mg of protein; activity with salicylaldehyde was2.8 nkat/mg.

[0298] Additional sequence analysis of DNA cloned from Ps. fluorescensAN103 was undertaken using cosmid clone pFI 793. The 1.8, 0.9 and 0.8 kbEcoRI/PstI fragments were sub-cloned into E. coli vector pUC18 and theirnucleotide sequences were determined. Sequencing the 0.9 kb sub-clonesrevealed that there are two different fragments of the same size. Thenucleotide sequences of DNA fragments of 1837 bp, 960 bp, 959 bp and 854bp in sub-clones pFI 901, pFI 912, pFI 913 and pFI 911 respectively arepresented in FIGS. 13 to 16. Outward reading PCR primers were designedfrom the ends of each of the four sequences as shown in FIG. 17. Use ofthese priners in all possible pairwise combination with pFI 793 astemplate showed that the 1.8 kb fragment of pFI 901 was separated fromthe 959 bp fragment of pFI 913 by 1.5 kb on the cosmid DNA (FIG. 18).Direct sequence analysis of this 1.5 kb PCR product enabled thistogether with the 1.8 kb and 959 bp fragments to be merged into onelarger fragment of 4.3 kb (FIG. 19).

EXAMPLE 6

[0299] Production of vanillin from trans-ferulovl SCoA and enzymeactivities II and III

[0300] Trans-feruloyl SCoA was synthesised as described in Example 2,and was used as a substrate of the trans-feruloyl SCoA hydratase/aldolcleavage enzyme (ie a single polypeptide with enzyme activities II andIII) as purified by the method described in Example 4. Vanillin wasproduced from trans-feruloyl SCoA.

EXAMPLE 7

[0301] A transgenic tobacco plant which produces vanillin

[0302]Nicotiana tabacum (tobacco) is transformed using a strain ofAgrobacterium tumefaciens which has been modified so that it transfersthe Ps. fluorescens gene encoding enzyme activities II and III (seeExample 5) to the tobacco plant. The tobacco plant produces vanillin inthose parts of the plant which have trans-feruloyl SCoA present, atleast in the form of a vanillin glycoside.

1. A method of producing vanillin comprising the steps of (1) providingtrans-ferulic acid or a salt thereof; and (2) providingtrans-ferulate:CoASH ligase activity (enzyme activity I), trans-feruloylSCoA hydratase activity (enzyme activity II), and4-hydroxy-3-methoxyphenyl-β-hydroxy-propionyl-S-CoA (HMPHP SCoA)cleavage activity (enzyme activity II).
 2. A method according to claim 1wherein means for converting vanillin to a non-vanillin product isabsent or reduced.
 3. A method according to claim 1 wherein the enzymeactivities I, II and III are provided by Pseudomonas fluorescens biovar.V, strain AN103 as deposited under the Budapest Treaty at the NationalCollections of Industrial and Marine Bacteria Limited, Scotland underAccession No NCIMB 40783, or a mutant or variant thereof.
 4. A methodaccording to any one of claims 1 to 3 wherein the enzyme activities I,II and III are provided by an intact-cell-free system of Pseudomonasfluorescens biovar. V, strain AN103 as deposited under the BudapestTreaty at the National Collections of Industrial and Marine BacteriaLimited, Scotland under Accession No NCIMB 40783, or a mutant or variantthereof.
 5. A method according to claim 2 wherein means for convertingvanillin to a non-vanillin product is an activity that interconvertsvanillin and vanillic acid (enzyme activity IV).
 6. A method accordingto claim 5 wherein NAD⁺ is absent.
 7. A method according to claim 1 or 2further comprising the step of (3) providing any one of the cofactorsCoenzyme ASH, ATP or Mg²⁺, or other functionally equivalent cofactors.8. A method according to claim 7 wherein either one of the cofactorsCoenzyme ASH and ATP is recycled or generated.
 9. A method according toclaim 8 wherein Coenzyme ASH is recycled using the enzymes citratesynthase and citrate lyase.
 10. A method according to claim 8 whereinATP is generated using the enzymes-adenylate kinase and acetate kinase.11. A method according to any one of claims 1 to 10 wherein thetrans-ferulic acid or salt thereof is provided by action oftrans-ferulic acid esterase on plant material, said plant materialcontaining an ester of trans-ferulic acid.
 12. A method according to anyone of claims 1 to 11 wherein at least one of the enzyme activities IIor III is provided by a substantially purified enzyme.
 13. A methodaccording to any one of claims 1 to 12 further comprising the step ofproviding a compound other than trans-ferulic acid or a salt thereofwhich may be converted by any one of enzyme activities I, II or III intoa desirable product.
 14. A method according to claim 13 wherein saidcompound is any of trans-4coumaric acid or a salt thereof,trans4-coumaroyl S CoA, trans-caffeic acid or a salt thereof,trans-caffeoyl SCoA or 3,4-methylene dioxy-trans-cimlamic acid or a saltthereof.
 15. A method according to claim 14 wherein said compound istrans4-coumaric acid or a salt thereof or trans4-coumaroyl SCoA.
 16. Amethod of producing vanillic acid, or a salt thereof, comprising thesteps as defined in any one of claims 1, 3, 4 and 7 to 12 and thefurther step of providing enzyme activity IV.
 17. A method according toany one of claims 1 to 12 comprising the further step of separatingvanillin from the other reaction components.
 18. A method according toclaim 16 comprising the further step of separating vanillic acid, or asalt thereof, from the other reaction components.
 19. Pseudomonasfluorescens biovar V, strain AN103 as deposited under the BudapestTreaty at the National Collections of Industrial and Marine BacteriaLimited, Scotland under Accession No NCIMB 40783, or a mutant or variantthereof.
 20. A mutant of Pseudomonas fluorescens biovar V, strain AN103according to claim 18 which accumulates vanillin when provided withtrans-ferulic acid or a salt thereof.
 21. A polypeptide which, in thepresence of appropriate cofactors if any, is capable of catalysing theinterconversion of trans-feruloyl S CoA and4-hydroxy-3-methoxyphenyl-β-hydroxy-propionyl SCoA (HMPHP SCoA).
 22. Apolypeptide according to claim 21 which is a trans-feruloyl S CoAhydratase.
 23. A polypeptide which, in the presence of appropriatecofactors if any, is capable of catalysing the interconversion of4-hydroxy-3-methoxyphenyl-β-hydroxy-propionyl SCoA (HMPHP SCoA) andvanillin.
 24. A polypeptide according to claim 23 which is a HMPHP SCoAcleavage enzyme.
 25. A polypeptide which, in the presence of appropriatecofactors if any, is capable of catalysing the interconversion oftrans-feruloyl-S-CoA and vanillin via HMPHP SCoA.
 26. A polypeptideaccording to any one of claims 21, 23 and 25 comprising the amino acidsequence MetSerThrTyrGluGlyArgTrpLysThrValLysVaLGluIleGluAspGlyILeAlaPheValIleLeuAsnArgProGluLysArgAsnAlaMetSerProThrLeuAsnArgGluMetIleAspValLeuGluThrLeuGluGlnAspProAlaAlaGlyValLeuValLeuThrGlyAlaGlyGluAlaTrpThrAlaGlyMetAspLeuLysGluTyrPheArgGluValAspAlaGlyProGluIleLeuGlnGluLysIleArgArgGluAlaSerGlnTrpGlnTrpLysLeuLeuArgMetTyrAlaLysProThrIleAlaMetValAsnGlyTrpCysPheGlyGlyGlyPheSerProLeuValAlaCysAspLeuAlaIleCysAlaAspGluAlaThrPheGlyLeuSerGluIleAsnTrpGlyIleProProGlyAsnLeuValSerLysAlaMetAlaAspThrValGlyHisArgGlnSerLeuTyrTyrIleMetThrGlyLysThrPheGlyGlyGlnLysAlaAlaGluMetGlyLeuValAsnGluSerValProLeuAlaGlnLeuArgGluValThrIleGluLeuAlaArgAsnLeuLeuGluLysAsnProValValLeuArgAlaAlaLysHisGlyPheLysArgCysArgGluLeuThrTrpGluGlnAsnGluAspTyrLeuTyrAlaLysLeuAspGlnSerArgLeuLeuAspThrGluGlyGlyArgGluGlyGlyMetLysGlnPheLeuAspAspLysSerIleLysProGlyLeuGlnAlaTyrLysArg

(SEQ ID No 2) or a fragment or variant thereof.
 27. A vanillin:NAD⁺oxidoreductase comprising the amino acid sequenceMetLeuAspValProLeuLeuIleGlyGlyGlnSerCysProAlaArgAspGlyArgThrPheGluArgArgAsnProValThrGlyGluLeuValSerArgValAlaAlaAlaThrLeuGluAspAlaAspAlaAlaValAlaAlaAlaGlnGlnAlaPheProAlaTrpAlaAlaLeuAlaProAsnGluArgArgSerArgLeuLeuLysAlaAlaGluGlnLeuGlnAlaArgSerGlyGluPheIleGluAlaAlaGlyGluThrGlyAlaMetAlaAsnTrpTyrGlyPheAsnValArgLeuAlaAlaAsnMetLeuArgGluAlaAlaSerMetThrThrGlnValAsnGlyGluValIleProSerAspValProGlySerPheAlaMetAlaLeuArgGlnProCysGlyValValLeuGlyIleAlaProTrpAsnAlaProValIleLeuAlaThrArgAlaIleAlaMetProLeuAlaCysGlyAsnThrValValLeuLysAlaSerGluLeuSerProAlaValHisArgLeuIleGlyGlnValLeuGlnAspAlaGlyLeuGlyAspGlyValValAsnValIleSerAsnAlaProAlaAspAlaAlaGlnIleValGluArgLeuIleAlaAsnProAlaValArgArgValAsnPheThrGlySerThrHisValGlyArgIleValGlyGluLeuSerAlaArgHisLeuLysProAlaLeuLeuGluLeuGlyGlyLysAlaProLeuLeuValLeuAspAspAlaAspLeuGluAlaAlaValGlnAlaAlaAlaPheGlyAlaTyrPheAsnGlnGlyGlnIleCysMetSerThrGluArgLeuIleValAspAlaLysValAlaAspAlaPheValAlaGlnLeuAlaAlaLysValGlyThrLeuArgAlaGlyAspProAlaAspProGlySerValLeuGlySerLeuValAspAlaSerAlaGlyThrArgIleLysAlaLeyIleAspAspAlaValAlaLysGlyAlaArgLeuValIleGlyGlyGlnLeuGluGlySerIleLeuGlnProThrLeuLeuAspGlyValAspAlaSerMetArgLeuTyrArgGluGluSerPheGlyProValAlaValValLeuArgGlyGlyGlyGluGluAlaLeuLeuGlnLeuAlaAsnAspSerGluPheGlyLeuSerAlaAlaIlePheSerArgAspThrGlyArgAlaLeuAlaLeuAlaGlnArgValGluSerGlyIleCysHisIleAsnGlyProThrValHisAspGluAlaGlnMetProPheGlyGlyValLysSerSerGlyTyrGlySerPheGlyGlyLysAlaSerIleGluHisPheThrGlnLeuArgTrpValThrLeuGlnAsnGlyProArgHisTyr ProIle

(SEQ ID No 4) or a fragment or variant thereof.
 28. A polypeptide asdefined in any one of claims 21 to 27 which is substantially pure.
 29. Apolynucleotide encoding a polypeptide as defined in any one of claims 21to 27 .
 30. A polynucleotide comprising at least a part of thePseudomonas fluorescens DNA contained within the cosmid clone pFI 793 asdeposited under the Budapest Treaty at the National Collections ofIndustrial and Marine Bacteria Limited. Scotland under Accession NoNCIMB 40777, or a fragment or variant thereof.
 31. Cosmid pFI 793 asdeposited under the Budapest Treaty at the National Collections ofIndustrial and Marine Bacteria Limited, Scotland under Accession NoNCIMB
 40779. 32. A polynucleotide according to claim 30 encoding apolypeptide as defined in any one of claims 21 to 27 .
 33. Apolynucleotide according to any one of claims 29, 30 or 32 comprisingthe nucleotide sequenceATGAGCACATACGAAGGTCGCTGGAAAACGGTCAAGGTCGAAATCGAAGACGGCATCGCGTTTGTCATCCTCAATCGCCCGGAAAAACGCAACGCGATGAGCCCGACCCTGAACCGCGAGATGATCGATGTTCTGGAAACCCTCGAGCAGGACCCTGCCGCCGGTGTGCTGGTGCTGACCGGTGCGGGCGAAGCCTGGACCGCAGGCATGGACCTCAAGGAATACTTCCGCGAAGTGGACGCCGGCCCGGAAATCCTCCAGGAAAAAATCCGCCGCGAAGCCTCGCAATGGCAATGGAAACTGCTGCGCATGTACGCCAAGCCGACCATCCCCATGGTCAATGGCTCGTGCTTCGGCGGCGGTTTCAGCCCGCTGGTGGCCTGCGACCTGGCGATCTTCGCCGACGAAGCAACCTTCGGTCTCTCGGAAATCAACTGGGGTATCCCGCCGGGCAACCTGGTGAGCAAGGCCATGGCCGACACCGTGGGCCACCGCCAGTCGCTCTACTACATCATGACCGGCAAGACCTTCGGTGGGCAGAAAGCCGCCGAGATGGGCCTGGTCAACGAAAGCGTGCCCCTGGCGCAACTGCGCGAACTCACCATCGAGCTGGCGCGTAACCTGCTCGAAAAAAACCCGGTGGTGCTGCGTGCCGCCAAACACGGTTTCAAACGCTGCCGCGAACTGACCTGGGAGCAGAACGAGGATTACCTGTACGCCAAGCTCGATCACTCCCCTTTGCTGGACACCGAAGGCGCTCGCGAGCAGGGCATGAAGCAATTCCTCGACGACAAGAGCATCAAGCCTGGCCTGCAAGCGTATAAAGCG,

(SEQ ID No 1) or a fragment or variant thereof.
 34. A polynucleotideaccording to any one of claims 29, 30 or 32 comprising the nucleotidesequence ATGCTGGACGTGCCCCTGCTGATTGGCGGCCAGTCGRGCCCCGCGCGCGACGGTCGAACCTTCGAGCGCCGCAACCCGGTGACTGGCGAGTTGGTGTCGCGGGTTGVVGCCGCCSCCCTGGAAGATGCCGACGCCGCCGTGGCCGCTGCCCAGCAAGCGTTTCCCGCGTGGGCCGCGCTGGCGCCCAATGAACGGCGCAGCCGTTTGCTCAAGGCCGCCGAACAATTGCAGGCGCGCAGCGGCGAGTTCATCGAGGCGGCGGGCGAGACCGGCGCCATGGCCAACTGGTACGGGTTCAACGTACGGCTGGCGGCCAACATGCTGCGTGAAGCGGCATCGATGACCACCCAGGTCAATGGTGAAGTGATTCCCTCGGACGTTCCCGGCAGTTTCGCCATGGCCCTGCGCCAGCCCTGTGGCGTGGTGCTGGGCATCGCCCCCTGGAACGCCCCGGTGATTCTCGCCACCCGGGCGATTGCCATGCCGCTGGCCTGTGGCAACACCGTGGTGCTGAAGGCTTCCGAGCTGAGTCCGGCGGTGCATCGCTTGATCGGCCAGGTGCTGCAGGACGCCGGCCTGGGCGATGGCGTGGTCAACGTCATCAGTAATGCGCCGGCGGATGCGGCACAGATTGTCGAGCGCCTGATTGCCAACCCGGCCGTACGCCGGGTCAATTTCACCGGTTCGACCCACGTCGGGCGCATTGTCGGCGAGCTCTCGGCGCGCCACCTCAAACCGGCGTTGCTCGAGCTGGGCGGCAAGGCACCGTTGCTGGTGCTCGACGATGCCGACCTGGAGGCTGCCGTGCAGGCGGCGGCGTTTGGCGCCTACTTCAACCAGGGACAGATCTGTATGTCCACCGAGCGCCTGATTGTCGATGCCAAGGTGGCCGACGCCTTTGTCGCCCAGTTGGCGGCCAAGGTCGAGACCCTGCGCGCCGGTGATCCTGCCGACCCGGAGTCGGTGCTCGGTTCGCTGGTGGACGCCAGCGCTGGCACGCGGATCAAAGCGTTGATCGATGATGCCGTGGCCAAGGGCGCGCGCCTGGTAATCGGCGGGCAACTGGAGGGCAGCATCTTGCAGCCGACCCTGCTCGACGGTGTCGACGCGAGCATGCGTTTGTACCGCGAAGAGTCCTTCGGCCCGGTGGCGGTGGTGCTGCGCGGCGAGGGCGAAGAAGCGCTGTTGCAACTGGCCAACGACTCCGAGTTCGGTTTGTCGGCGGCGATTTTCAGTCGTGACACCGGCCGTGCCCTGGCCCTGGCCCAGCGGGTCGAATCGGGCATCTGCCACATCAACGGCCCGACCGTGCACGACGAAGCGCAAATGCCTTTTGGCGGGGTCAAGTCCAGCGGCTACGGCAGTTTTGGCGGCAAGGCATCGATTGAGCATTTCACTCAGTTGCGCTGGGTCACCCTCCAGAATGGTCCACGGCACTAT CCGATC

(SEQ ID No 3) or a fragment or variant thereof.
 35. A nucleic acidvector comprising a polynucleotide according to any one of claims 28 to34 .
 36. A host cell comprising a polynucleotide as defined in any oneof claims 28 to 34 .
 37. A host cell according to claim 36 comprisingnucleic acid which encodes any one of the polypeptides as defined inclaims 21, 23 and
 27. 38. A host cell according to claim 36 or 37 whichis a bacterium or yeast.
 39. A host cell according to claim 38 which isa food-grade bacterium or yeast.
 40. A host cell according to claim 39which is a Lactococcus spp. or a Lactobacillus spp. or Saccharomycescerevisiae or a biovar thereof.
 41. A host cell according to claim 37which is a plant cell.
 42. A host cell according to claim 41 whereinsaid plant cell is a cell from any one of Nicotiana spp., Solanumtuberosum, Brassica spp., Beta spp., Capsicum spp. and Vanilla spp. 43.A host cell according to claim 41 or 42 wherein said cell is comprisedin a plant.
 44. A transgenic plant comprising at least onepolynucleotide according to claim 29 and which, as a consequence of thepresence of said polynucleotide, expresses any of the enzyme activitiesII and III.
 45. A transgenic plant according to claim 45 which, as aconsequence of the presence of said polynucleotides, expresses theenzyme activities II and III.
 46. A transgenic plant according to claim44 or 45 wherein said plant is selected from Nicotiana spp., Solanumtuberosum, Brassica spp., Beta spp., Capsicum spp. and Vanilla spp. 47.A method according to claims 1 to 18 wherein the enzyme activities IIand III are provided by the host cell according to any of claims 36 to43 or a transgenic plant according to any one of claims 44 to 46 , or anextract thereof.
 48. A method according to claims 1 to 18 wherein theenzyme activities I, II and III are provided by a microorganism whichcan convert trans-ferulic acid to vanillin.
 49. Use of Pseudomonasfluorescens biovar. V, strain AN103 or a mutant or derivative thereoffor producing vanillin, or vanillic acid or salt thereof.
 50. Use of apolypeptide according to any one of claims 21 to 28 for producingvanillin, or vanillic acid or salt thereof.
 51. Use of a polynucleotideaccording to any one of claims 29 to 35 for producing vanillin, orvanillic acid or salt thereof.
 52. Use of a host cell according to anyone of claims 36 to 43 for producing vanillin, or vanillic acid or saltthereof.
 53. Use of a transgenic plant according to any one of claims 44to 46 for producing vanillin, or vanillic acid or salt thereof.
 54. Useof Pseudomonas fluorescens biovar V, strain AN103 or a mutant orderivative thereof for converting a compound, other than trans-ferulicacid or a salt thereof, into a desirable product.
 55. Use of apolypeptide according to any one of claims 21 to 28 for converting acompound into a desirable product.
 56. Use of a polynucleotide accordingto any one of claims 29 to 35 for converting a compound into a desirableproduct.
 57. Use of a host cell according to any one of claims 36 to 43for converting a compound into a desirable product.
 58. Use of atransgenic plant according to any one of claims 44 to 46 for convertinga compound, other than trans-ferulic acid or a salt thereof, into adesirable product.
 59. Use according to any one of claims 54 to 58wherein said desirable product is a flavour or aroma.
 60. Use accordingto claims 54 to 59 wherein said compound is any one of trans4-coumaroylS CoA and trans-caffeoyl SCoA.
 61. A food or beverage comprising a hostcell according to any one of claims 36 to 43 , or an extract thereof.62. A food or beverage comprising a transgenic plant according to claim44 to 46, or a part or extract thereof.
 63. A method of producingvanillin comprising (1) providing trans-feruloyl SCoA; and (2) providingtrans-feruloyl SCoA hydratase activity (enzyme activity II), and HMPHPSCoA clevage activity (enzyme activity III).
 64. Any novel method ofproducing vanillin or vanillic acid substantially as disclosed herein.65. Any novel polypeptide substantially as disclosed herein.
 66. Anynovel polynucleotide substantially as disclosed herein.